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

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journal contribution
posted on 27.04.2016, 00:00 by Seyed R. Tabaei, Feng Guo, Florentine U. Rutaganira, Setareh Vafaei, Ingrid Choong, Kevan M. Shokat, Jeffrey S. Glenn, Nam-Joon Cho
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)­P2 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)­P2 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 EC50 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.