Structure-function study of CDK complexes

The growth and division of cells is strictly controlled at the molecular level by a series of enzymes that include the cyclin dependent protein kinases (CDKs). These enzymes are switched on and off in an orderly sequence to ensure that cell division starts and stops at the required time. Though closely related in sequence, biological studies of the different members of the CDK family have revealed that each has unique properties. Our work, using the technique of X-ray crystallography, allows us to see the structure of these molecules at atomic resolution and so to learn how they differ from each other. The first aim of our work is to identify the sequence patterns that characterise substrates of the CDK family. We can use these patterns to identify potential substrates encoded within the human genome. These studies will also identify substrates suitable for analysis by X-ray crystallography that will provide an understanding of how they interact with CDKs. CDK activity is regulated by CDK binding to members of the cyclin and CKS protein families. The second aim of our work is to use kinetic, biophysical and structural methods to provide explanations for how cyclins and CKS proteins regulate CDK activity. Aberrant CDK activity has been linked to cancer, neurological diseases, and rheumatoid arthritis. In recent years, understanding a particular defect that leads to disease has led to exciting new medicines directed towards a particular target (e.g. the drugs Gleevec, Iressa and Herceptin for cancer treatment). A number of CDK-selective inhibitors are in clinical trials for the treatment of cancer. These agents all act by binding to the CDK active site to block CDK activity. Compounds that block other interactions made by CDK/cyclin complexes represent an alternative target for CDK-directed therapies. The work described in this project, to elaborate the interactions that mediate the binding of substrates to CDKs and CDK-regulatory proteins, will aid the further development of such compounds and may also reveal additional targets for inhibitor development.

Cyclin-dependent protein kinases (CDKs) are implicated in many cellular regulatory processes, notably in the control of cell cycle progression and transcription. By definition, CDK activity requires association with an activating cyclin molecule, with many family members further activated by phosphorylation of a conserved threonine residue within the activation loop (Thr160 in CDK2). While CDK1 is the only cell-cycle regulatory CDK that appears to be essential for cell viability and development in multicellular organisms, knockout of other CDK family members gives rise to phenotypes that suggest family members may have specific roles associated with the phosphorylation of a unique set of substrates. The first aim of this project is to identify novel CDK1, CDK2 and CDK4-specific substrates by screening an unbiased cDNA library using an approach that has been previously used to identify CDK2 substrates. The second aim of this project is to further elaborate the structural basis of CDK substrate selectivity in CDK/cyclin and CDK/cyclin/regulator complexes. Specifically we will (i) employ screening and biochemical methods to characterise and compare the substrate selectivity of CDK1/, and CDK2/, and CDK4/cyclin complexes; (ii) establish how CDK1 selectivity is modulated by interaction with the Cdc28 kinase subunit regulators Cks1 and Cks2; (iii) elaborate the role of the cyclin B recruitment site in determining CDK1 substrate selectivity; and (iv) use X-ray crystallography to define in atomic detail the molecular interactions that dictate the substrate selectivity of CDK 1, 2 and 4 complexes identified through our screening (objectives i and ii) and biochemical/biophysical (objective iii) studies. Structure determination and functional characterisation of these various complexes will contribute to our further understanding of control of cell cycle progression and cell division.