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Anal Chem. 2016 May 17;88(10):5042-5. doi: 10.1021/acs.analchem.6b01293. Epub 2016 Apr 28.

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

Author information

1
School of Materials Science and Engineering and Centre for Biomimetic Sensor Science, Nanyang Technological University , 50 Nanyang Avenue, 639798, Singapore.
2
Departments of Medicine, Division of Gastroenterology and Hepatology, and Microbiology & Immunology, Stanford University School of Medicine , Stanford, California 94305, United States.
3
Howard Hughes Medical Institute, Department of Cellular and Molecular Pharmacology, University of California San Francisco , San Francisco, California 94158-2280, United States.
4
Palo Alto Veterans Administration Medical Center , Palo Alto, California 94304, United States.

Abstract

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.

PMID:
27118725
PMCID:
PMC5291064
DOI:
10.1021/acs.analchem.6b01293
[Indexed for MEDLINE]
Free PMC Article

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