Array-based fluorescence assay for serine/threonine kinases using specific chemical reaction.
We report herein the development of an efficient fluorescence assay for serine/threonine kinases using a peptide array. Our approach is based on chemical reactions specific to phosphoserine and phosphothreonine residues, that is, base-mediated beta-elimination of the phosphate group and subsequent Michael addition of a thiol-containing fluorescent reagent. This procedure enables the covalent introduction of a fluorescent moiety into the phosphorylated peptide. Novel fluorescent reagents were designed for this purpose and synthesized. With these reagents, protein kinase A (PKA) and Akt-1 activities were readily detected. Our method can also be used to measure the activity of kinase inhibitors. This assay is expected to be widely applicable in kinase research.
Akita
Shoji
S
Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe-dori, Mizuho-ku, Nagoya, Aichi 467-8603, Japan.
Umezawa
Naoki
N
Kato
Nobuki
N
Higuchi
Tsunehiko
T
D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry*; D007700:Kinetics; D010455:Peptides / Q000737:chemistry; D010750:Phosphoproteins / Q000737:chemistry; D010766:Phosphorylation; D010768:Phosphoserine / Q000737:chemistry; D010769:Phosphothreonine / Q000737:chemistry; D040081:Protein Array Analysis / Q000379:methods*; D047428:Protein Kinase Inhibitors / Q000737:chemistry; D017346:Protein Serine-Threonine Kinases / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000378:metabolism
D005456:Fluorescent Dyes; D010455:Peptides; D010750:Phosphoproteins; D047428:Protein Kinase Inhibitors; D010769:Phosphothreonine; D010768:Phosphoserine; D017346:Protein Serine-Threonine Kinases