Development of an assay to screen for inhibitors of tau phosphorylation by cdk5

• Published in: Journal of biomolecular screening Vol. 9; no. 2; pp. 122 - 131
• Main Authors: Ahn, Jae Suk, Musacchio, Andrea, Mapelli, Marina, Ni, Jake, Scinto, Leonard, Stein, Ross, Kosik, Kenneth S, Yeh, Li-An
• Format: Journal Article
• Language: English
• Published: United States 01.03.2004
• Subjects: Adenosine Triphosphate - analysis; Adenosine Triphosphate - metabolism; Antibodies, Monoclonal - metabolism; Binding Sites; Biotinylation; Cells, Cultured; Cyclin-Dependent Kinase 5; Cyclin-Dependent Kinases - metabolism; Humans; Kinetics; Kinetin; Miniaturization; Phosphorylation; Protein Binding; Purines - antagonists & inhibitors; Recombinant Proteins - metabolism; Substrate Specificity; tau Proteins - metabolism; Time Factors
• ISSN: 1087-0571; 2472-5552
• DOI: 10.1177/1087057103260594

A high-throughput assay for tau phosphorylation by cdk5/p25 is described. Full-length recombinant tau was used as a substrate in the presence of saturating adenosine triphosphate (ATP). Using PHF-1, an antibody directed specifically against 2 tau phosphorylation epitopes (serine 396 and serine 404), an enzyme-linked immunosorbent assay (ELISA)-based colorimetric assay was formatted in 384-well plates. The assay was validated by measuring kinetic parameters for cdk5/p25 catalysis and known inhibitors. Rate constants for the site-specific phosphorylations at the PHF-1 epitopes were determined and suggested preferential phosphorylation at these sites. The performance of this assay in a high-throughput format was demonstrated and used to identify inhibitors of tau phosphorylation at specific epitopes phosphorylated by cdk5/p25.