CDK-containing macromolecular assemblies

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The behaviour of a cell is defined by the set of genes it expresses and by its commitment to either a quiescent or a proliferating state. The cyclin-dependent kinases (CDKs) bind to non-catalytic cyclin subunits to form CDK-cyclin complexes that play central roles in regulating both gene-expression and cell-proliferation. Consequently, CDK-cyclins are important in normal cells and in a variety of disease conditions. We are proposing a programme of research to understand how CDK-cyclins work, how they in turn are regulated, and how their inappropriate activity can contribute to the development of disease.Because certain CDK-cyclin complexes can function interchangeably, expression of all but one cell-cycle regulatory CDK can be inhibited without compromising the cells ability to divide. The exception is CDK1, which must possess unique properties that make it indispensable. We have solved the structure of CDK1-containing complexes, and found that it has a small number of unique kinetic and structural properties. We propose to use mutagenesis to engineer these properties into CDK2, the closest homologue of CDK1, and see if the resulting CDK2-variant can functionally complement CDK1. In this way, we will identify what is the property of CDK1 that explains its uniqueness, resolving a key conundrum at the heart of CDK-biology.CDK1 and CDK2 play important roles in meiosis, the process by which sperm and egg cells are generated. These CDKs can be regulated by binding of non-cyclin partners and by covalent modifications other than phosphorylation. We will explore how these regulatory mechanisms work, and how conventional and unconventional regulatory mechanisms operate in meiosis.Different CDK-cyclin complexes are characterised as regulating either transcription (e.g. CDK9-cyclin T) or proliferation (e.g. CDK4-cyclin D). A growing body of evidence, however, suggests that this classification is over simplified. In particular cyclin D appears to work together with nuclear hormone receptors (such as the androgen and oestrogen receptors) to regulate transcription, and we will investigate the mechanisms that underlie this. Because inhibition of androgen receptor signalling is a well validated way to combat prostate cancer, a better understanding of how cyclin D contributes to androgen receptor function may suggest ways to tackle forms of prostate cancer that have become resistant to current therapies.Several transcriptional and cell-cycle regulatory CDKs depend on a chaperone system to shepherd them into the correct shape and/or correct set of complexes to carry out their cellular roles. The chaperoning of CDKs is mediated by HSP90, a promiscuous chaperone, and by Cdc37, a more specific adaptor. We have reconstituted the process of "hand-off" from chaperone to functional CDK-cyclin complexes in cell-free experiments. We now propose to characterise the structural mechanism of hand off, using a combination of biophysical and structural techniques.X-ray crystallography has provided a structural explanation of how cyclins both activate CDKs and, to some extent, direct them towards appropriate substrates. For CDK9-cyclin T, a further level of regulation is provided by interchange between two settings: an inhibitory particle termed the 7SK ribonucleoprotein (7SKsnRNP) and an activated setting, where CDK9-cyclin T is recruited to genes that are being expressed. Recent technical developments make large complexes such as the 7SKsnRNP accessible to high resolution structural study by cryoelectron microscopy, and we will use this technique to image the 7SKsnRNP to understand the mechanism by which it holds CDK9-cyclin T inactive, and how it subsequently switches into the activated setting.