THE OPTIMIZATION OF A TIME-RESOLVED, VESICLE-BASED FLUORESCENCE ASSAY FOR THE ACTIVITY OF THE LIPID KINASE PI4K-IIIBETA AND THE EFFECT OF LIPID TRANSFER PROTEINS ON ENZYME ACTIVITY
type: CreativeWork
description: Human PI4K-IIIβ is an 89 kDa phosphatidylinositol (PI) kinase that phosphorylates its substrate headgroup at position C-4, thus producing PI(4)P. This phosphoinositide is the most abundant in the trans-Golgi network where it is essential for secretory vesicle formation, as well as the precursor for other phosphoinositides that are crucial for intracellular signalling. Among others, phosphoinositide homeostasis in eukaryotic membranes rely on PI kinases and PI transfer proteins (PITPs). In yeast, the PITP Sec14p is known to exchange PI and phosphatidylcholine between lipid bilayers in vitro and proposed to present PI to be phosphorylated by the PI4-kinase Pik1 in a heterotypic ligand exchange fashion. However, the precise mechanism by which this interaction occurs has yet to be elucidated. To explore how and if PITPs and PI4K-IIIβ work as hypothesized, we expressed and purified recombinant human PI4K-IIIβ in Escherichia coli and assayed lipid kinase activity using an optimized real-time, vesicle-based fluorescence assay. After comparing different affinity tags, deletion mutants and expressing cell lines, GST-tagged wild-type PI4K-IIIβ was chosen and expressed in Rosetta 2(DE3) cells with a 2.5-fold increase in the native protein yield when compared to other methods. Proteins were further purified by an addition heat shock protein removal wash. The resulting PI4K-IIIβ displayed activity comparable to the commercially available, insect cell expressed counterpart. Optimization of the activity assay afforded a robust assay that displayed protein concentration dependent response while using unilamellar liposomes as the substrate. Agreeing with previous reports, the activity of PI4K-IIIβ was greatly reduced by wortmannin and increased by Triton X-100. The activity of PI4K-IIIβ was tested in the presence of active human PITPα and PITPβ, as well as yeast Frequenin and Sec14p, but none of them elicited a reproducible enhancement on PI(4)P production by PI4K-IIIβ. A similar pa…
abstract: Human PI4K-IIIβ is an 89 kDa phosphatidylinositol (PI) kinase that phosphorylates its substrate headgroup at position C-4, thus producing PI(4)P. This phosphoinositide is the most abundant in the trans-Golgi network where it is essential for secretory vesicle formation, as well as the precursor for other phosphoinositides that are crucial for intracellular signalling. Among others, phosphoinositide homeostasis in eukaryotic membranes rely on PI kinases and PI transfer proteins (PITPs). In yeast, the PITP Sec14p is known to exchange PI and phosphatidylcholine between lipid bilayers in vitro and proposed to present PI to be phosphorylated by the PI4-kinase Pik1 in a heterotypic ligand exchange fashion. However, the precise mechanism by which this interaction occurs has yet to be elucidated. To explore how and if PITPs and PI4K-IIIβ work as hypothesized, we expressed and purified recombinant human PI4K-IIIβ in Escherichia coli and assayed lipid kinase activity using an optimized real-time, vesicle-based fluorescence assay. After comparing different affinity tags, deletion mutants and expressing cell lines, GST-tagged wild-type PI4K-IIIβ was chosen and expressed in Rosetta 2(DE3) cells with a 2.5-fold increase in the native protein yield when compared to other methods. Proteins were further purified by an addition heat shock protein removal wash. The resulting PI4K-IIIβ displayed activity comparable to the commercially available, insect cell expressed counterpart. Optimization of the activity assay afforded a robust assay that displayed protein concentration dependent response while using unilamellar liposomes as the substrate. Agreeing with previous reports, the activity of PI4K-IIIβ was greatly reduced by wortmannin and increased by Triton X-100. The activity of PI4K-IIIβ was tested in the presence of active human PITPα and PITPβ, as well as yeast Frequenin and Sec14p, but none of them elicited a reproducible enhancement on PI(4)P production by PI4K-IIIβ. A similar pa…
author: Muniz Correa, Marcelo Victor
language: eng
keywords: Heterotypic lipid exchange, PI4K-IIIbeta, Sec14, kinase activity assay, phosphoinositides
license: http://creativecommons.org/publicdomain/zero/1.0/
date published: 2023-09-14T15:38:27Z
date modified: 2026-07-24T01:23:00Z
provider: Brock University
publisher: Networked Digital Library of Theses and Dissertations
type: WebPage
source organization: Brock University

PropertyValue
property ID: Handle
value: http://hdl.handle.net/10464/18092