TY - JOUR
T1 - Multistep Compositional Remodeling of Supported Lipid Membranes by Interfacially Active Phosphatidylinositol Kinases
AU - Tabaei, Seyed R
AU - Guo, Feng
AU - Rutaganira, Florentine U.
AU - Vafaei, Setareh
AU - Choong, Ingrid
AU - Shokat, Kevan M.
AU - Glenn, Jeffrey S.
AU - Cho, Nam-Joon
PY - 2016/5/17
Y1 - 2016/5/17
N2 - 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.
AB - 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.
U2 - 10.1021/acs.analchem.6b01293
DO - 10.1021/acs.analchem.6b01293
M3 - Article
SN - 0003-2700
VL - 88
SP - 5042
EP - 5045
JO - Analytical Chemistry
JF - Analytical Chemistry
IS - 10
ER -