Related Experiment Video
Updated: Oct 13, 2025

Experimental Approaches to Study Mitochondrial Localization and Function of a Nuclear Cell Cycle Kinase, Cdk1
Published on: February 25, 2016
Reconstitution and use of highly active human CDK1:Cyclin-B:CKS1 complexes
Pim J Huis In 't Veld1, Sabine Wohlgemuth1, Carolin Koerner1
1Department of Mechanistic Cell Biology, Max Planck Institute of Molecular Physiology, Dortmund, Germany.
Abstract:
As dividing cells transition into mitosis, hundreds of proteins are phosphorylated by a complex of cyclin-dependent kinase 1 (CDK1) and Cyclin-B, often at multiple sites. CDK1:Cyclin-B phosphorylation patterns alter conformations, interaction partners, and enzymatic activities of target proteins and need to be recapitulated in vitro for the structural and functional characterization of the mitotic protein machinery. This requires a pure and active recombinant kinase complex. The kinase activity of CDK1 critically depends on the phosphorylation of a Threonine residue in its activation loop by a CDK1-activating kinase (CAK). We developed protocols to activate CDK1:Cyclin-B either in vitro with purified CAKs or in insect cells through CDK-CAK co-expression. To boost kinase processivity, we reconstituted a ternary complex consisting of CDK1, Cyclin-B, and CKS1. In this work, we provide and compare detailed protocols to obtain and use highly active CDK1:Cyclin-B (CC) and CDK1:Cyclin-B:CKS1 (CCC).
More Related Videos
12:26Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay
Published on: May 3, 2018
10:33Development of Inhibitors of Protein-protein Interactions through REPLACE: Application to the Design and Development Non-ATP Competitive CDK Inhibitors
Published on: October 26, 2015
Related Concept Videos
Positive Regulator Molecules
Inhibition of Cdk Activity
M-Cdk Drives Transition Into Mitosis
Cyclin-dependent kinases, or Cdks, work in concert with cyclins to control cell cycle transitions. M-Cdk, a complex of Cdk1 bound to M cyclin, is a well-known example of this coordinated control that drives the transition from the G2 to the M phase.
M cyclin...
Anaphase Promoting Complex
S-Cdk Initiates DNA Replication
Two states at the origin of replication
In eukaryotes, the initiation of replication occurs at many sites on the chromosomes, called the origins of...
Mitogens and the Cell Cycle