HTScan^® CDK1/CycB Kinase Assay Kit

The GST-Kinase fusion protein was produced using a baculovirus expression system with a construct expressing full-length human CDK1 (Met1-Met295) (GenBank Accession No. NM_001786) and full-length human CycB (Met1-Val433) (GenBank Accession No. NM_031966), both with an amino-terminal GST tag. The protein was purified by one-step affinity chromatography using glutathione-agarose.

The kit provides a means of performing kinase activity assays with recombinant human CDK1/CycB kinase. It includes active CDK1/CycB kinase (supplied as a GST fusion protein), a biotinylated peptide substrate and a phospho-serine/threonine antibody for detection of the phosphorylated form of the substrate peptide.

Cyclins and cyclin-dependent kinases (CDK) are key regulators in mammalian cell cycle. Regulation of these complexes occurs through cyclin production and its destruction, relocation, inhibitory/activating phoshorylation, relocation and modification by other proteins. Each cyclin associates with one or two CDKs and most CDKs associate with one or two cyclins (1-3). CDK1 forms a complex with cyclin A/B and regulates phosphorylation of cytoskeleton proteins involved in mitosis. CDK2 and CDK3 form complexes with cyclin E, which regulate the G1-S phase transition while the CDK2/CycA complex regulates S phase progression (4,5). CDK4/CycD and CDK6/CycD are activated by mitogenic signaling during early G1 and progressively accumulate as cells transition through this phase of the cell cycle. CDK5 is activated in postmitotic neurons and regulates neuron migration during brain development (6). CDK7/CycH is believed to form a link between transcription and cell cycle. CDK8/CycC and CDK9/CycT are involved in transcription (1,2). The kinase activity of CDKs is tightly regulated by phosphorylation and protein-protein interactions. Activation of CDKs requires binding to a specific cyclin and phosphorylation of a conserved threonine residue in a region called the T loop. Examining the phosphorylation of peptides by CDK/cyclin complexes suggests that both CDKs and cyclins play a role in recognizing substrates. A consensus sequence, (S/T)PX(R/K), is identified in the peptides that are phosphorylated by CDK/cyclins.