Development of an assay to screen for inhibitors of tau phosphorylation by cdk5.
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.
Center for Neurologic Diseases, Brigham and Women's Hospital, Harvard Medical School, Boston, USA.
Jae Suk
JS
Ahn
Andrea
A
Musacchio
Marina
M
Mapelli
Jake
J
Ni
Leonard
L
Scinto
Ross
R
Stein
Kenneth S
KS
Kosik
Li-An
LA
Yeh
D000255:Adenosine Triphosphate / Q000032:analysis / Q000378:metabolism; D000911:Antibodies, Monoclonal / Q000378:metabolism; D001665:Binding Sites; D019921:Biotinylation; D002478:Cells, Cultured; D051360:Cyclin-Dependent Kinase 5; D018844:Cyclin-Dependent Kinases / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D007701:Kinetin; D008904:Miniaturization; D010766:Phosphorylation; D011485:Protein Binding; D011687:Purines / Q000037:antagonists & inhibitors; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity; D013997:Time Factors; D016875:tau Proteins / Q000378:metabolism*
D000911:Antibodies, Monoclonal; C085349:PHF-1 monoclonal antibody; D011687:Purines; D011994:Recombinant Proteins; D016875:tau Proteins; C090046:olomoucine; D000255:Adenosine Triphosphate; D051360:Cyclin-Dependent Kinase 5; C495905:CDK5 protein, human; D018844:Cyclin-Dependent Kinases; D007701:Kinetin