Analysis of the regulation and function of the mitotic kinase Citron kinase in cell division

Cells are the building blocks of many organisms, including humans. Growth, development and reproduction in all these organisms depend on the accurate and fascinating process of cell division, which faithfully partitions the genetic information between the two dividing cells. Proper cell division is also crucial for determining cell fate and tissue organization. Errors during this process are responsible for many genetic diseases, including Down's syndrome, microcephaly, sterility, and cancer. Thus, a thorough understanding of the mechanisms that control cell division may lead to the development of novel therapeutic treatments for these genetic diseases. Moreover, recent evidence also indicates a clear link between cell division and ageing and therefore a knowledge of the cell division process can help understand both the natural process of ageing and the origin of early ageing-related diseases.

Complex control and surveillance mechanisms have evolved to ensure the fidelity and robustness of cell division. The genetic material - DNA - is compacted into chromosomes during cell division and is not accessible for the production of new factors. Therefore, the mechanisms that control cell division rely in large part on the regulation of the function and activity of proteins that have been generated before cells begin dividing. One class of proteins that play a key role in controlling the accuracy and fidelity of cell division are the serine/threonine kinases. These kinases can regulate the function, dynamics and activity of numerous other cell division proteins, known as substrates, by adding phosphate groups - a process know as phosphorylation - that alter the mechanical and structural properties of their targets. The goal of this project is to dissect the role of one of this kinase, Citron kinase (CIT-K), which controls different aspects of cell division. We propose to use cutting edge post-genomic technologies to identify all the substrates of CIT-K throughout cell division and to understand how CIT-K is itself regulated through phosphorylation by other kinases. As CIT-K has been linked to human primary microcephaly and proposed as a potential target in anti-cancer therapy, our research will not only help understand key mechanisms that control cell division and proliferation, but will also lay the foundation for the development of future therapies for the treatment of these human pathologies.

The fundamental process of cell division requires the coordinated action of many proteins that control complex and yet finely regulated events. As during cell division the DNA is packed into chromosomes, all these events are regulated by post-translational modifications, including phosphorylation and protein degradation. Therefore, cell division represents also a unique and powerful model system to study the function, activity and regulation of kinases and their counteracting phosphatases.

Here we propose to investigate in detail the regulation and function of the mitotic kinases Citron kinase (CIT-K), which has been neglected and misunderstood for many years and only recently shown to function throughout mitosis and linked to human disease. We plan to use a combination of chemical genetics and quantitative phospho-proteomics to identify the substrates of CIT-K and study the role of its kinase activity at different stages of mitosis. We will also study the regulation of CIT-K by two other mitotic kinases, Aurora B and Cdk1, by mutating the CIT-K residues phosphorylated by these two kinases into phospho-mimetic and phospho-dead amino acids. These mutants will then be employed in a series of in vivo and in vitro assays to elucidate the role of these phosphorylations events on the function, dynamics and activity of CIT-K during cell division, including its interaction with other mitotic proteins and cellular components. Finally, we will create bioinformatics tools that will allow comparative and integrated analysis of the protein-protein interaction networks of CIT-K and other mitotic proteins in different conditions, with the ultimate goal of providing the scientific community with a useful and versatile bioinformatics platform for the mining and analysis of phosphorylation-mediated regulation of protein-protein interaction networks in cell division.

Our research will benefit three sectors:

1) Academic community:
The research proposed in this application will improve our knowledge of the mechanisms that control cytokinesis and contribute to the scientific advancement in the fields of cell division and cancer and benefit the academic community worldwide. We will disseminate our findings to the scientific community by attending national and international scientific conferences and present our research in form of poster or oral communications. We will publish our results in the form of research articles or reviews in peer-reviewed scientific journal and make our publications widely accessible by publishing in open access journals or by depositing our articles in PubMed Central. We will also generate several reagents and data that will be useful to the academic community, including cDNAs and constructs, antibodies, and transgenic and gene-edited human cell lines. In addition, the proteomic data will be entered in a database that allows systematic comparison and analysis of MS data and correlation with other datasets. This database will be very valuable for the academic community. Moreover, during the duration of the grant we will train 1 postdoctoral research associate, 1 graduate student and at least 3 undergraduate students. The postdoc will be directly employed from this grant, while the PI has been promised a Departmental PhD studentship for this project.

2) Business/Industry:
Mutations in Citron kinase (CIT-K) have been implicated in human primary microcephaly and CIT-K has been proposed as anti-cancer target. Thus, the research proposed here could lead to the identification of new reagents and to new knowledge that could aid in the diagnosis of human primary microcephaly and the treatment of cancer pathologies. For example some of our phospho-specific antibodies might become useful biomarkers. Moreover, the antibodies and cell lines generated during this project could also have commercial value. To exploit, patent and license any possible commercial applications of our research, we will consult with Cambridge Enterprise Ltd, a wholly owned subsidiary of the University of Cambridge responsible for the commercialisation of technology arising from the University's research. Cambridge Enterprises has established a programme of scientific open collaboration with GlaxoSmithKline. In addition the PI is a member of the Cambridge Cancer centre, which organises annual meetings with AstraZeneca to discuss cancer research in Cambridge and its potential therapeutic and commercial exploitations.

3) General public:
Our findings could lead to potential novel therapeutic treatments for human pathologies, such as cancer and microcephaly. We could for example identify new biomarkers that could aid in the identification and prognosis of these pathologies and this would have a major impact for the health and well being of people worldwide.
Our research will also provide novel insights into the mechanisms that control cell division. Cell division is a basic process essential for normal growth, development, reproduction, and aging in humans and thus our results will directly benefit the culture of the public in the UK.
To communicate our research and engage with the public, we will participate to some of the activities organised by the Office of External Affairs and Communications (OEAC) of the University of Cambridge, including:
-The University of Cambridge Science Festival organised every year by the OEAC;
-School Roadshow organised by the OAEC in parallel with the Science festival;
-Public lectures organised by the Department of Pathology charity CAMPOD;
-Communication with mass media in synergy with the Communications Team of the OEAC, which handles publicity and media enquiries on behalf of the University of Cambridge.