Related Experiment Video
Updated: Aug 15, 2026
Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay
Published on: May 3, 2018
Measurement of cdk4 kinase activity using an affinity peptide-tagging technology
J J Wu1, D R Yarwood, M A Sills
1Novartis Institute for Biomedical Research, Novartis Pharmaceuticals Corporation, Summit, NJ 07901, USA. jinzi.wu@phrma.novartis.com
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.
Insights
A new homogeneous assay using affinity peptide-tagging technology enables rapid discovery of Cdk4 inhibitors. This robust method simplifies screening by eliminating separation steps, aiding cancer research.
Area of Science:
- Biochemistry
- Molecular Biology
- Cancer Research
Background:
- Cyclin-dependent kinases (CDKs), including CDK4, regulate cell cycle progression.
- CDK4 misregulation is linked to various cancers, highlighting the need for targeted inhibitors.
- Developing efficient screening assays for CDK4 inhibitors is crucial for cancer therapy.
Purpose of the Study:
- To develop a novel homogeneous assay for rapid discovery of CDK4 inhibitors.
- To utilize affinity peptide-tagging technology for simplified kinase assays.
- To establish a robust and high-throughput screening method for CDK4 inhibitors.
Main Methods:
- Engineered a fusion protein (GST-Rb152-StrepTag) with a retinoblastoma protein domain and a StrepTag.
- Used the engineered protein as a substrate for in vitro CDK4 kinase assays.
- Employed scintillation proximity assay (SPA) with streptavidin-coated beads for signal detection.
Main Results:
- The homogeneous assay successfully detected CDK4 phosphorylation activity.
- Affinity peptide tagging eliminated the need for separation and wash steps, simplifying the assay.
- The assay demonstrated robustness and suitability for high-throughput screening.
Conclusions:
- Developed a simple, robust, and homogeneous assay for CDK4 inhibitor screening.
- Affinity peptide-tagging technology offers a generic platform for kinase and protein-interaction assays.
- This method accelerates the discovery of potential anti-cancer therapeutics targeting CDK4.
