Interplay between the Polo and Scant/Greatwall mitotic kinases

Summary of Public Engagement in Science
The process of cell division has been highly conserved throughout evolution. This means that simple cells like yeasts use many of the same molecules to regulate cell division as do our own cells. The process whereby enzymes called protein kinases modify cellular proteins though addition of phosphates is commonly used to change the functions of proteins as cells proceed through their division cycles. This was first demonstrated by the Nobel Prize winning finding that the major cell cycle protein kinase, the cyclin dependent kinase, is so highly conserved that the human enzyme will rescue a deficiency of the corresponding enzyme in yeast cells. We use the fruit-fly, Drosophila, in our research because the cells of this multi-cellular organism have a division apparatus that closely resembles that found in human cells. Some 15 years ago we discovered two protein kinases of Drosophila, termed Polo and Aurora, that regulate the function of this division apparatus. We now know that elevated expression of the human counterparts of these two enzymes contributes to the development of tumours. Indeed inhibitors of these molecules are now being developed by pharmaceutical companies for use in cancer therapy. The present research aims for a better understanding of the roles of not only Polo kinase but also a novel mitotic kinase, Scant/Greatwall, that appears to participate in common processes as Polo. To this end we continue to use the fruit-fly as a model firstly because it offers sophisticated genetic tools for studying regulatory pathways. Secondly we are able to follow the behaviour of its division apparatus in living cells in order to study the effects of interfering with the function of individual proteins. Our findings can be rapidly translated to the regulation of cell division in human tumour cells. We anticipate that the work will lead to finding new proteins that regulate the cell division cycle, several of which might be valid targets for the future development of anti-cancer agents. In any event, an improved knowledge of the underlying biology behind the cell division process will contribute to the rational design of new agents to combat proliferative cell disease.

Originally discovered in Drosophila, the mitotic Polo-like and Aurora-like kinases are overexpressed in human tumours and have become recognised as potential drug targets. The Drosophila gene Scant was discovered by a dominant mutation that results in defects of mitotic spindle poles but only when the dose of Polo kinase is halved. We have reverted the dominant ScantD mutation to generate recessive alleles with mitotic phenotypes that suggest roles in the fidelity of repair of DNA damage and in metaphase-anaphase progression. We propose to study interactions between the ScantD-polo combination and members of the DNA damage sensing pathway in Drosophila known to influence centrosome behaviour. Through screens for enhancers and suppressors of the ScantD-polo interaction we will also move towards understanding the pathway it represents. We have identified Scant as an allele of greatwall (gwl). We will employ both genetic and molecular approaches to identify proteins with which the Scant/Gwl protein kinase interacts. We will study mitosis and meiosis in an allelic series of Scant/gwl mutants employing both immunostaining of fixed preparations and time-lapse studies on live cells to study kinetochore behaviour, chromosome congression, chromatid separation/segregation and destruction of APC/C substrates. Cek1, the fission yeast counterpart of Scant/Gwl, acts on the same pathway as Cut8, a proposed regulator of proteasome function. We will therefore monitor Cut8 and proteasome behaviour in Scant/gwl mutants and characterise the phenotypes of Drosophila cut8 mutants. We have found a novel phosphoprotein Endos that interacts with a proteasome component. Mutations in endos result in a phenotype similar to gwl. We will map the phosphorylation sites on the Endos protein and study the consequences of mutations in such sites on mitotic progression. A number of tagging approaches will be used to purify proteins that interact with Endos. We will apply similar techniques to analyse molecular interactions of the Polo and Scant/Gwl protein kinases. We will study the mitotic effects of down-regulation of three protein kinases related to Scant/Gwl in the human genome and screen for their possible involvement in oncogenesis. Characterisation of the regulation of the Polo, Scant/Gwl and their related mitotic kinases will ultimately lead to the development of more rational approaches to the use of their inhibitors for cancer therapy and also to identification of additional drug targets.