A high-performance liquid chromatography assay for Dyrk1a, a Down syndrome-associated kinase.

Abstract

Down syndrome is the most common aneuploidy. It is caused by the presence of an extra copy of chromosome 21. Several studies indicate that aberrant expression of the kinase Dyrk1a (dual-specificity tyrosine phosphorylation-regulated kinase 1a) is implicated in Down syndrome, in particular in the onset of mental retardation. Moreover, elevated Dyrk1a activity may also be a risk factor for other neurodegenerative disorders such as Alzheimer's disease. Over the past years, Dyrk1a has appeared as a potential drug target. Availability of sensitive and quantitative enzyme assays is of prime importance to understand the role of Dyrk1a and to develop specific inhibitors. Here, we describe a new method to measure Dyrk1a activity based on the separation and quantification of specific fluorescent peptides (substrate and phosphorylated product) by high-performance liquid chromatography (HPLC). Kinetic and mechanistic analyses using well-known inhibitors of Dyrk1a confirmed the reliability of this approach. In addition, this assay was further validated using brain extracts of mice models expressing different copies of the Dyrk1a gene. Our results indicate that this novel Dyrk1a assay is simple, sensitive, and specific. It avoids the use of radioactivity-based approaches that, until now, have been widely employed to measure Dyrk1a activity.

• Authors: Linh C Bui, Laure Tabouy, Florent Busi, Jean-Marie Dupret, Nathalie Janel, Chris Planque, Jean-Maurice Delabar, Fernando Rodrigues-Lima, Julien Dairou
• Journal: Analytical biochemistry (Anal Biochem) Vol. 449 Pg. 172-8 (Mar 15 2014) ISSN: 1096-0309 [Electronic] United States
• PMID: 24374000 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
• Copyright: Copyright © 2013 Elsevier Inc. All rights reserved.

Chemical References

• Fluorescent Dyes
• Peptides
• Dyrk kinase
• Protein-Tyrosine Kinases
• Protein Serine-Threonine Kinases
• Fluorescein

Topics

• Amino Acid Sequence
• Animals
• Brain (enzymology)
• Chromatography, High Pressure Liquid (methods)
• Down Syndrome (enzymology)
• Enzyme Assays (methods)
• Fluorescein (analysis)
• Fluorescence
• Fluorescent Dyes (analysis)
• Humans
• Mice
• Molecular Sequence Data
• Peptides (analysis, metabolism)
• Phosphorylation
• Protein Serine-Threonine Kinases (analysis, metabolism)
• Protein-Tyrosine Kinases (analysis, metabolism)
• Reproducibility of Results