ac6b01293_si_001.pdf (1.11 MB)
Multistep Compositional Remodeling of Supported Lipid Membranes by Interfacially Active Phosphatidylinositol Kinases
journal contribution
posted on 2016-04-27, 00:00 authored by Seyed
R. Tabaei, Feng Guo, Florentine U. Rutaganira, Setareh Vafaei, Ingrid Choong, Kevan M. Shokat, Jeffrey S. Glenn, Nam-Joon ChoThe
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.