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
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 pattern was observed with the human PI3-kinase, PIK3C3. Our results demonstrate that a PI presentation model based on heterotypic exchange may not occur in vitro, suggesting either that PITPs’ role in phosphoinositide production could rely uniquely on maintaining sufficient PI pools in the Golgi membrane or that additional protein partners may be required for the regulation of PI4K-IIIβ by PITPs.
Heterotypic lipid exchange, PI4K-IIIbeta, Sec14, kinase activity assay, phosphoinositides
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