Chemical biology in a Cdk network

Cyclin-dependent kinases (Cdks) are essential for the control of cell cycle transitions, and changes in activity of a single Cdk1-cyclin B complex can order the cell cycle in fission yeast. Using mathematical modelling and experiments in Xenopus egg extracts, we have shown that it is not Cdk1 activity per se , that determines the cell cycle, but the ratio of activities of Cdk1 and the Cdk1-counteracting phosphatase, PP2A. Inhibiting PP2A can inappropriately promote mitotic onset even if Cdk1 activity is very low. Thus, it is essential to consider the kinetic properties of the phosphorylation network in order to understand how a single kinase complex can generate an ordered cell cycle with abrupt transitions. To better understand the redundancy of Cdk control of the cell cycle, we have undertaken an integrated chemical biology approach. Mathematical modelling of enzymology data predicted conditions allowing selective chemical Cdk2 inhibition. Together with experiments using Xenopus egg extracts, this supports a rate-limiting role for Cdk2 in DNA replication. To confirm this, we designed inhibitor-resistant (ir)-Cdk2 mutants using a novel bioinformatics approach. Bypassing Cdk2 inhibition with ir-Cdk2 mutants or with Cdk1 demonstrates that Cdk2 is indeed rate-limiting for DNA replication, but increasing Cdk1 activity can compensate for inhibition of Cdk2. Additionally, we crystalised wild-type and ir-mutant Cdk2 with the Cdk2-selective inhibitor NU6102 and, for the first time, the Cdk1-selective inhibitor RO-3306. Crystal structures revealed alternative binding modes of these inhibitors, and mechanisms of Cdk2 inhibitor resistance. This work thus provides insights into structure, functions and biochemistry of Cdks, which has important implications for Cdk inhibitor use in cancer treatment. Posters selected for Short Oral Presentation