title: 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.affiliation: Unit of Functional and Adaptative Biology (BFA), Université Paris Diderot, Sorbonne Paris Cité, CNRS UMR 8251, 75013 Paris, France.
authors.forename: Linh C
authors.initials: LC
authors.lastname: Bui
authors.affiliation: Unit of Functional and Adaptative Biology (BFA), Université Paris Diderot, Sorbonne Paris Cité, CNRS UMR 8251, 75013 Paris, France.
authors.forename: Laure
authors.initials: L
authors.lastname: Tabouy
authors.affiliation: Unit of Functional and Adaptative Biology (BFA), Université Paris Diderot, Sorbonne Paris Cité, CNRS UMR 8251, 75013 Paris, France.
authors.forename: Florent
authors.initials: F
authors.lastname: Busi
authors.affiliation: Unit of Functional and Adaptative Biology (BFA), Université Paris Diderot, Sorbonne Paris Cité, CNRS UMR 8251, 75013 Paris, France.
authors.forename: Jean-Marie
authors.initials: JM
authors.lastname: Dupret
authors.affiliation: Unit of Functional and Adaptative Biology (BFA), Université Paris Diderot, Sorbonne Paris Cité, CNRS UMR 8251, 75013 Paris, France.
authors.forename: Nathalie
authors.initials: N
authors.lastname: Janel
authors.affiliation: Unit of Functional and Adaptative Biology (BFA), Université Paris Diderot, Sorbonne Paris Cité, CNRS UMR 8251, 75013 Paris, France.
authors.forename: Chris
authors.initials: C
authors.lastname: Planque
authors.affiliation: Unit of Functional and Adaptative Biology (BFA), Université Paris Diderot, Sorbonne Paris Cité, CNRS UMR 8251, 75013 Paris, France.
authors.forename: Jean-Maurice
authors.initials: JM
authors.lastname: Delabar
authors.affiliation: Unit of Functional and Adaptative Biology (BFA), Université Paris Diderot, Sorbonne Paris Cité, CNRS UMR 8251, 75013 Paris, France. Electronic address: fernando.rodrigues-lima@univ-paris-diderot.fr.
authors.forename: Fernando
authors.initials: F
authors.lastname: Rodrigues-Lima
authors.affiliation: Unit of Functional and Adaptative Biology (BFA), Université Paris Diderot, Sorbonne Paris Cité, CNRS UMR 8251, 75013 Paris, France. Electronic address: julien.dairou@univ-paris-diderot.fr.
authors.forename: Julien
authors.initials: J
authors.lastname: Dairou
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000201:enzymology; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004314:Down Syndrome / Q000201:enzymology*; D057075:Enzyme Assays / Q000379:methods*; D019793:Fluorescein / Q000032:analysis; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000032:analysis; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D010455:Peptides / Q000032:analysis / Q000378:metabolism; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000032:analysis* / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000032:analysis* / Q000378:metabolism; D015203:Reproducibility of Results; D000097603:Dyrk Kinases
chemical_list: D019793:Fluorescein; D005456:Fluorescent Dyes; D010455:Peptides; D017346:Protein Serine-Threonine Kinases; D011505:Protein-Tyrosine Kinases; D000097603:Dyrk Kinases
keywords: Down syndrome; Dyrk1a; Enzyme assay; HPLC; Kinase