Multistep Compositional Remodeling of Supported Lipid Membranes by Interfacially Active Phosphatidylinositol Kinases


The multienzyme catalytic phosphorylation of phosphatidylinositol (PI) in a supported lipid membrane platform is demonstrated for the first time. One-step treatment with PI 4-kinase IIIβ (PI4Kβ) yielded PI 4-phosphate (PI4P), while a multistep enzymatic cascade of PI4Kβ followed by PIP 5-kinase produced PI-4,5-bisphosphate (PI­(4,5)­P<sub>2</sub> or PIP2). By employing quartz crystal microbalance with dissipation monitoring, we were able to track membrane association of kinase enzymes for the first time as well as detect PI4P and PI­(4,5)­P<sub>2</sub> generation based on subsequent antibody binding to the supported lipid bilayers. Pharmacologic inhibition of PI4Kβ by a small molecule inhibitor was also quantitatively assessed, yielding an EC<sub>50</sub> value that agrees well with conventional biochemical readout. Taken together, the development of a PI-containing supported membrane platform coupled with surface-sensitive measurement techniques for kinase studies opens the door to exploring the rich biochemistry and pharmacological targeting of membrane-associated phosphoinositides

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oai:figshare.com:article/3204583Last time updated on 2/12/2018

This paper was published in FigShare.

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