Development of an Assay to Screen for Inhibitors of Tau Phosphorylation by Cdk5-Reference-Cited by-同舟云学术

Development of an Assay to Screen for Inhibitors of Tau Phosphorylation by Cdk5

Published:2004-03 Issue:2 Volume:9 Page:122-131
ISSN:1087-0571
Container-title:Journal of Biomolecular Screening
language:en
Short-container-title:J Biomol Screen

Author:

Ahn Jae Suk¹,Musacchio Andrea²,Mapelli Marina²,Ni Jake³,Scinto Leonard⁴,Stein Ross³,Kosik Kenneth S.⁵,Yeh Li-An³

Affiliation:

1. Center for Neurologic Diseases, Brigham and Women’s Hospital, Harvard Medical School, Boston

2. Department of Experimental Oncology, European Institute of Oncology, Milan, Italy

3. Laboratory for Drug Discovery in Neurodegeneration, Brigham and Women s Hospital, Harvard Medical School, Boston

4. Laboratory for Higher Cortical Functions, Department of Neurology, Brigham and Women s Hospital, Harvard Medical School, Boston

5. Center for Neurologic Diseases, Brigham and Women s Hospital, Harvard Medical School, Boston

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. ( Journal of Biomolecular Screening 2004:122-131)

Publisher

Elsevier BV

Link

http://journals.sagepub.com/doi/pdf/10.1177/1087057103260594

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