title: A high-throughput liposome substrate assay with automated lipid extraction process for PI 3-kinase.
abstract: The signaling pathways involving lipid kinase class I phosphatidylinositol 3-kinases (PI 3-kinases) regulate cell growth, proliferation, and survival. Class I PI 3-kinases catalyze the conversion of PI (4,5)P(2) to PI (3,4,5)P(3), which acts as a lipid second messenger to activate mitogenic signaling cascades. Recently, p110alpha, a class IA PI 3-kinase, was found to be mutated frequently in many human cancers. Therefore, it is increasingly studied as an anticancer drug target. Traditionally, PI 3-kinase activities have been studied using liposome substrates. This method, however, is hampered significantly by the labor-intensive manual lipid extraction followed by a low-throughput thin-layer chromatography analysis. The authors describe a high-throughput liposome substrate-based assay based on an automated lipid extraction method that allows them to study PI 3-kinase enzyme mechanism and quantitatively measure inhibitor activity using liposome substrates in a high-throughput mode. This improved assay format can easily be extended to study other classes of phosphoinositide lipid kinases.
authors.affiliation: Millennium Pharmaceuticals, Inc., Cambridge, Massachusetts 02139, USA.
authors.forename: Trupti
authors.initials: T
authors.lastname: Lingaraj
authors.forename: John
authors.initials: J
authors.lastname: Donovan
authors.forename: Zhi
authors.initials: Z
authors.lastname: Li
authors.forename: Ping
authors.initials: P
authors.lastname: Li
authors.forename: Amanda
authors.initials: A
authors.lastname: Doucette
authors.forename: Sean
authors.initials: S
authors.lastname: Harrison
authors.forename: Jeffrey A
authors.initials: JA
authors.lastname: Ecsedy
authors.forename: Lenny
authors.initials: L
authors.lastname: Dang
authors.forename: Wenhai
authors.initials: W
authors.lastname: Zhang
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry; D001331:Automation; D001671:Biochemistry / Q000379:methods; D002855:Chromatography, Thin Layer / Q000379:methods; D006801:Humans; D020128:Inhibitory Concentration 50; D007700:Kinetics; D008055:Lipids / Q000737:chemistry; D008081:Liposomes / Q000737:chemistry*; D009369:Neoplasms / Q000175:diagnosis* / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000737:chemistry* / Q000378:metabolism; D010766:Phosphorylation; D015398:Signal Transduction; D013379:Substrate Specificity; D013997:Time Factors
chemical_list: D008055:Lipids; D008081:Liposomes; D000255:Adenosine Triphosphate; D019869:Phosphatidylinositol 3-Kinases