A high-throughput liposome substrate assay with automated lipid extraction process for PI 3-kinase.
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.
Lingaraj
Trupti
T
Millennium Pharmaceuticals, Inc., Cambridge, Massachusetts 02139, USA.
Donovan
John
J
Li
Zhi
Z
Li
Ping
P
Doucette
Amanda
A
Harrison
Sean
S
Ecsedy
Jeffrey A
JA
Dang
Lenny
L
Zhang
Wenhai
W
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
D008055:Lipids; D008081:Liposomes; D000255:Adenosine Triphosphate; D019869:Phosphatidylinositol 3-Kinases