Measurement of Cdk4 Kinase Activity Using an Affinity Peptide-Tagging Technology

Authors: Wu J.J.; Yarwood D.R.; Chaudhuri B.; Muller L.; Zurini M.; Sills M.A.

Source: Combinatorial Chemistry & High Throughput Screening, Volume 3, Number 1, February 2000 , pp. 27-36(10)

Publisher: Bentham Science Publishers

Abstract:

Cyclin dependent kinases such as Cdk4 are involved in the control of cell cycle progression, and misregulation of Cdk4 has been implicated in many types of cancers. In the present study, we report the development of a novel homogeneous assay using an affinity peptide-tagging technology for rapidly discovering Cdk4 inhibitors. The DNA sequence encoding a streptavidin recognition motif, or StrepTag (AWRHPQFGG), was cloned and expressed at the C-terminus of a fusion protein of a 152-amino acid hyperphosphorylation domain (Rb152) of the retinoblastoma protein (Rb) linked to GST at the N-terminus. This affinity peptide-tagged protein (GST-Rb152-StrepTag), which contains the two known phosphorylation sites of Rb, specifically phosphorylated by Cdk4 in vivo, was used as a substrate in the current in vitro kinase assay. After phosphorylation, scintillation proximity assay (SPA) scintillant beads coated with streptavidin were added. Radiolabeled GST-Rb152-StrepTag was brought in close proximity to the SPA scintillant beads through the interaction between StrepTag and streptavidin, resulting in the emission of light from beads. By applying the affinity peptide-tagging technology, we have eliminated the separation and wash steps which are normally required in a radioactive filtration assay. Therefore, this homogeneous method is simple, robust, and highly amenable to high-throughput screening of Cdk4-specific inhibitors. Furthermore, the affinity peptide tagging technique reported here is a simple, generic method that can be applied to many recombinant proteins for the development of kinase and protein-protein interaction assays.

Keywords: Cdk4 kinase actiity; Strep tag; yclin dependent kinases; Scintillation proximity assay; Filter binding assay; Phosphorylation; GST Rb152; Biotinylated peptide

Language: English

Document Type: Regular paper

DOI: http://dx.doi.org/10.2174/1386207003327774

Publication date: 2000-02-01

• Combinatorial Chemistry & High Throughput Screening publishes full length original research articles and reviews describing various topics in combinatorial chemistry (e.g. small molecules, peptide, nucleic acid or phage display libraries) and/or high throughput screening (e.g. developmental, practical or theoretical). Ancillary subjects of key importance, such as robotics and informatics, will also be covered by the journal. In these respective subject areas, Combinatorial Chemistry & High Throughput Screening is intended to function as the most comprehensive and up-to-date medium available. The journal should be of value to individuals engaged in the process of drug discoveryand development, in the settings of industry, academia or government.

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• By this author: Wu J.J. ;  Yarwood D.R. ;  Chaudhuri B. ;  Muller L. ;  Zurini M. ;  Sills M.A.

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