Measurement of Cdk4 kinase activity using an affinity peptide-tagging technology

Cyclin-dependent kinases such as Cdk4 are involved in the control of cell c ycle progression, and misregulation of Cdk4 has been implicated in many typ es of cancers. In the present study, we report the development of a novel h omogeneous assay using an affinity peptide-tagging technology for rapidly d iscovering Cdk4 inhibitors. The DNA sequence encoding a streptavidin recogn ition motif, or StrepTag (AWRHPQFGG), was cloned and expressed at the C-ter minus 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 t he two known phosphorylation sites of Rb, specifically phosphorylated by Cd k4 in vivo, was used as a substrate in the current in vitro kinase assay. A fter phosphorylation, scintillation proximity assay (SPA) scintillant beads coated with streptavidin were added. Radiolabeled GST-Rb152-StrepTag was b rought in close proximity to the SPA scintillant beads through the interact ion between StrepTag and streptavidin, resulting in the emission of light f rom beads. By applying the affinity peptide-tagging technology, we have eli minated the separation and wash steps which are normally required in a radi oactive filtration assay. Therefore, this homogeneous method is simple, rob ust; and highly amenable to high-throughput screening of Cdk4-specific inhi bitors. Furthermore, the affinity peptide tagging technique reported here i s a simple, generic method that can be applied to many recombinant proteins for the development of kinase and protein-protein interaction assays.