The cell cycle is controlled by coordinated post-translational modifications, especially phosphorylation and ubiquitination. Cyclin dependent protein kinases (CDKs) are fundamental cell cycle regulators whose importance is conserved throughout the eukaryotes. CDKs act as ”molecular switches”, necessary to induce rapid and unidirectional transitions in the cell cycle, including the initiation of DNA replication and mitosis. The impact upon biomedical research of the discovery of CDKs was recognised by the award of the Nobel Prize in Medecine in 2001 to Nurse, Hartwell and Hunt. The challenge is now to understand the molecular circuitry upstream and downstream of these regulators.
CDK activation is the primary step that commits a cell to DNA replication and passage through the cell cycle. We found in the model system of fission yeast (Fisher and Nurse, 1996) that oscillation in activity of a single cyclin-dependent kinase complex suffices to order the eukaryotic cell-cycle. These results led to a quantitative model of CDK-mediated cell-cycle control, the principles of which are the same in vertebrates. In this model, high CDK activity promotes mitosis and prevents replication origin licensing, and lower CDK activity promotes S-phase. However, usually different cyclin-CDK complexes trigger DNA replication (S-phase CDK) and mitosis (M-phase CDK), but they may be functionally redundant, with absence of one cyclin or CDK being compensated for by another. This functional redundancy is still not well understood, and it has complicated our comprehension of the functions of individual CDKs.
One of our goals is to describe essential CDK targets and how their phosphorylation triggers and organises DNA replication. We would also like to understand how the CDK control network ensures that DNA replication and mitosis are ordered and never overlap, which would be extremely detrimental to the genome. And we want to know how the decision to replicate is made: ie how the upstream signals which trigger cell proliferation affect the CDK control network, and how the cell nucleus is reorganised from a quiescent state to a replication competent-state. To answer these questions we are applying a "systems biology" approach of functional proteomics.
The fundamental principles of cell cycle control are conserved in all eukaryotic cells, and most of our advances in understanding have come from the study of simple cell cycle models, such as yeast and eggs of amphibians. In our lab, the model system we use is that of Xenopus egg extracts, which autonomously undergo regulated cell-cycles in vitro, in the complete absence of transcription. We also use different human primary cells and cell lines to study conservation of mechanisms, enabling us to identify the most important.
Finally, because of their importance in controlling cell proliferation, CDKs are implicated in a wide variety of pathologies, including cancers, viral and parasitic infections, chronic inflammation, and even neural accidents or degenerative diseases. They are therefore attractive therapeutic targets, and many small-molecule CDK inhibitors have been developed. Chemical inhibition is also an important approach complementary to genetic knockdowns or knockouts for physiological studies, since it maintains the presence of all proteins studied and thereby avoids the formation of unnatural complexes. Using a combination of depletion and chemical inhibition approaches we recently found that CDK1 and CDK2 have distinct but redundant roles in promotion of replication origin firing in Xenopus egg extracts (Krasinska et al., 2008). We are now developing a novel chemical-genetic approach to precisely inhibit and restore functions of individual CDK-cyclin complexes.
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