Development of an assay to screen for inhibitors of tau phosphorylation by Cdk5

Jae Suk Ahn; Andrea Musacchio; Marina Mapelli; Jake Ni; Leonard Scinto; Ross Stein; Kenneth S. Kosik; Li An Yeh

doi:10.1177/1087057103260594

Development of an assay to screen for inhibitors of tau phosphorylation by Cdk5

Jae Suk Ahn, Andrea Musacchio, Marina Mapelli, Jake Ni, Leonard Scinto, Ross Stein, Kenneth S. Kosik, Li An Yeh

Istituto Europeo di Oncologia

Research output: Contribution to journal › Article › peer-review

Abstract

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.

Original language	English
Pages (from-to)	122-131
Number of pages	10
Journal	Journal of Biomolecular Screening
Volume	9
Issue number	2
DOIs	https://doi.org/10.1177/1087057103260594
Publication status	Published - Mar 2004

Keywords

Alzheimer's disease
Cdk5
HTS
Tau phosphorylation

ASJC Scopus subject areas

Analytical Chemistry
Clinical Biochemistry
Biotechnology
Biochemistry
Molecular Biology

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abstract = "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.",

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AU - Ahn, Jae Suk

AU - Musacchio, Andrea

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AU - Ni, Jake

AU - Scinto, Leonard

AU - Stein, Ross

AU - Kosik, Kenneth S.

AU - Yeh, Li An

PY - 2004/3

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KW - Alzheimer's disease

KW - Cdk5

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Development of an assay to screen for inhibitors of tau phosphorylation by Cdk5

Abstract

Keywords

ASJC Scopus subject areas

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