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)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.
School of Materials Science and Engineering and Centre for Biomimetic Sensor Science, Nanyang Technological University , 50 Nanyang Avenue, 639798, Singapore.
Seyed R
SR
Tabaei
Departments of Medicine, Division of Gastroenterology and Hepatology, and Microbiology & Immunology, Stanford University School of Medicine , Stanford, California 94305, United States.
Feng
F
Guo
Howard Hughes Medical Institute, Department of Cellular and Molecular Pharmacology, University of California San Francisco , San Francisco, California 94158-2280, United States.
Florentine U
FU
Rutaganira
School of Materials Science and Engineering and Centre for Biomimetic Sensor Science, Nanyang Technological University , 50 Nanyang Avenue, 639798, Singapore.
Setareh
S
Vafaei
Departments of Medicine, Division of Gastroenterology and Hepatology, and Microbiology & Immunology, Stanford University School of Medicine , Stanford, California 94305, United States.
Ingrid
I
Choong
Howard Hughes Medical Institute, Department of Cellular and Molecular Pharmacology, University of California San Francisco , San Francisco, California 94158-2280, United States.
Kevan M
KM
Shokat
Departments of Medicine, Division of Gastroenterology and Hepatology, and Microbiology & Immunology, Stanford University School of Medicine , Stanford, California 94305, United States.|Palo Alto Veterans Administration Medical Center , Palo Alto, California 94304, United States.
Jeffrey S
JS
Glenn
School of Materials Science and Engineering and Centre for Biomimetic Sensor Science, Nanyang Technological University , 50 Nanyang Avenue, 639798, Singapore.
Nam-Joon
NJ
Cho
D019870:1-Phosphatidylinositol 4-Kinase / Q000037:antagonists & inhibitors / Q000737:chemistry*; D004791:Enzyme Inhibitors / Q000737:chemistry; D008051:Lipid Bilayers / Q000737:chemistry*; D008565:Membrane Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry*; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000737:chemistry; D018129:Phosphatidylinositol Phosphates / Q000737:chemistry; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry*; D058525:Quartz Crystal Microbalance Techniques
D004791:Enzyme Inhibitors; D008051:Lipid Bilayers; D008565:Membrane Proteins; D019269:Phosphatidylinositol 4,5-Diphosphate; D018129:Phosphatidylinositol Phosphates; C037178:phosphatidylinositol 4-phosphate; D017853:Phosphotransferases (Alcohol Group Acceptor); D019870:1-Phosphatidylinositol 4-Kinase; C020077:1-phosphatidylinositol-4-phosphate 5-kinase