CDK-containing macromolecular assemblies

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.

This programme will exploit our collaborations and strengths in CDK reagent generation, biochemical/biophysical assay, target structural biology to illuminate the mechanisms that underlie essential roles of CDKs in regulating the cell cycle, meiosis, and transcription.

Through collaborations with Professor Mary Herbert and Professor Angel Nebrada, and building on structural studies of CDK1- and CDK2-containing complexes, we will investigate the roles of CDKs 1 and 2, as regulated by either canonical (cyclin A, cyclin B, cyclin E) or non-canonical (SPDY/RINGO) cyclin regulators, in meiosis and mitosis.

We have dissected a site of direct contact between CDK2-cyclin A and Skp1-Skp2. We will complete the characterization of this site and implement cellular studies to explore its role in the regulation of the localization and stability of p27, and of SKP2 itself (via interactions with CDH1).

Cyclin E can play a driver role in cancer. Through collaboration with the target validation group at the NICR, we will identify the properties of cyclin E that enable this role, suggesting mechanisms to target in anti-cancer drug discovery.

We hypothesize that cyclin D forms complexes that regulate transcription by a mosaic of interactions involving multiple peptide motifs binding to corresponding docking sites. Some of these sites are established but we have observed and/or inferred others in structural studies. We will characterize these interactions in cell-free systems, and test their relevance to (e.g.) AR-dependent transcription and proliferation in cellular systems through collaboration with Dr Luke Gaughan.

We will use biochemical and biophysical tools to study the interactions of P-TEFb, a key regulator of transcription, with its positive and negative regulators. In collaboration with Professor Hans Elmlund, we will solve high resolution cryoelectron microscopy structures for various states of the 7SK snRNP, a complex that sequesters and inhibits P-TEFb.

The major impact of our research will come from subsequent translational research informed by better understanding of CDK function. In addition, we expect to generate impact by ways that include:

i) Training a cohort of skilled researchers in interdisciplinary structural biology
Macromolecular crystallography (MX) is an essential enabling technology for the cellular and molecular biosciences, and consequently for UK pharmaceutical and biotechnological industries. The biotechnological and pharmaceutical industry that is dependent on this discipline contributes hugely to the UK economy: in 2010 the pharmaceutical sector provided 67,000 jobs, each contributing £195,000 of GVA, with 25,000 of these positions being in high skill R&D activities (source: http://www.abpi.org.uk). Use of MX in UK pharma/biotech requires a cohort of skilled practitioners, trained in interdisciplinary structural biology and with an awareness of the potential for contribution to medical and biotechnological practice. Support of this proposal will allow the Noble and Endicott groups to continue their contribution in this area.

ii) Building capacity in a cryoelectron microscopy
RCUK has recognized cryo EM as an important area for development, following recent exciting technical advances in the field. The proposed collaborative EM research will build UK presence and international networks in this area and will train a named researcher in the relevant experimental and computational techniques.

iii) Identifying important interactions to be targeted in the development of personalized medicine by commercial and/or academic drug discoverers
CDKs are the end effectors of pathways that function aberrantly in diseases ranging from HIV-AIDS (CDK9), through cardiac hypertrophy (CDK9) to cancer (CDK9, CDK4/6, CDK2). Accordingly, there are CDK inhibitors in preclinical development and/or clinical trials in each of these indications. These inhibitors all target the ATP-binding site. While this strategy has demonstrated utility, it does have drawbacks relating to selectivity and the potential for resistant mutations. This proposal will characterize the complexes in which CDKs participate, identifying protein:protein interactions that are essential to CDK function. Such interactions are increasingly being targeted for drug discovery, being often more amenable to selective inhibition. Particularly promising in this respect are cyclin-transcription factor interactions, such as cyclin D binding to the androgen receptor. Understanding these interactions in AR-driven proliferation may suggest new strategies to overcome castrate-resistant prostate cancer.

iv) Generating assays and reagents to enable the study of CDKs in diverse biological contexts
While study of the CDKs in regulating cell-cycle, transcription, cancer and meiosis is the focus of this programme, CDKs are implicated in other biological processes and diseases. The phosph-CDK2-cyclin A generating reagents and protocols that we developed have been widely distributed by JE to academic and commercial groups and commercialized by New England Biolabs. Similar impact can be expected from the proposed programme.

v) Participating in national and international networks
Networks provide a vehicle for disseminating results, know-how and practice that contribute broadly to the community. MN and JE will share outputs of the proposed programme through key networks that promote anticancer drug discovery, and structural biology software development.

vi) Inspiring and educating biomedical undergraduates in basic and applied research
The research will be conducted in the NICR, which contributes to Newcastle's undergraduate lecture courses, and hosts undergraduate and Masters students through 2-3 month projects. In both of these undertakings, the students will be exposed to the cutting-edge research supported by this grant, to the mutual benefit of researchers and students.