Real-time fluorogenic kinase assay using protein as substrate.
A real-time fluorogenic kinase assay using myelin basic protein (MBP) as a substrate is reported. MBP is part of a noncovalent complex with a negatively charged, dye-labeled lipopeptide, (N-heptadecanoyl)-K(dye2)-linker-EEIYGEF-amide. The complex is approximately 20 times less fluorescent than the free lipopeptide. The MBP-lipopeptide complex serves as a protein substrate for several Ser/Thr kinases. We infer that the observed fluorescence increase on the addition of kinase and ATP is due to the phosphorylation of MBP, which decreases the affinity of MBP with the negatively charged, dye-labeled lipopeptide. Several protein kinases (protein kinase C betaII, mitogen-activated protein kinase [MAPK] Erk1, and MAPK Erk2) were tested with the assay. The assay exhibited a fivefold fluorescence increase over background, provided kinetic values comparable to literature values (apparent K(m)(ATP)), and produced inhibitor constants comparable to literature values for a typical inhibitor, namely staurosporine.
Sun
Hongye
H
Life Technologies Corporation, Foster City, CA 94404, USA. hongye.sun@lifetech.com
Mitchell
Karen
K
Lee
Linda
L
D000595:Amino Acid Sequence; D057075:Enzyme Assays / Q000379:methods*; D005456:Fluorescent Dyes / Q000378:metabolism*; D020128:Inhibitory Concentration 50; D055666:Lipopeptides / Q000737:chemistry / Q000378:metabolism; D013058:Mass Spectrometry; D004676:Myelin Basic Protein / Q000378:metabolism*; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011494:Protein Kinases / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013997:Time Factors
D005456:Fluorescent Dyes; D055666:Lipopeptides; D004676:Myelin Basic Protein; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases