Regulation of Mps1, a protein kinase required for the spindle assembly checkpoint.

When cells divide, the genome must be copied prior to its equal partitioning to two new cells. Errors that occur during DNA segregation are passed on to progeny cells, and an absence or excess of certain gene products leads to serious diseases, such as cancer. To prevent unequal DNA distribution, cells have evolved a checkpoint mechanism in which enzymes called protein kinases monitor the arrangement and alignment of DNA in the cell, and inhibit DNA separation until all the chromosomes have been accounted for and aligned at the spindle. The protein kinase Mps1 has an important, but undefined, role in the checkpoint of all mammalian cells. Mps1 activity can be turned on or off by an unknown regulatory circuit in the cell. This study aims to identify how Mps1 activity is regulated. Several components of the checkpoint are implicated in cancer, and so molecules that directly target these proteins hold enormous promise as anti-cancer agents. A focused biochemical approach, using model cellular systems, is urgently required to facilitate an analysis of this poorly characterised checkpoint component. Importantly, these studies will generate experimental tools to investigate Mps1 function in normal cell division and in cancer cells.

Alarmingly, one in three people will be diagnosed with cancer during their lifetime, and in 2003 almost 300,000 new cancer cases were reported in the UK. One of the major cytological differences between normal cells and tumour cells is the abnormal chromosome complement found in the latter, suggestive of a direct link between aneuploidy and cancer. Moreover, it is now clear that aneuploid cells are produced due to chromosome missegregation during cell division. The process of chromosome segregation is monitored and regulated by the spindle assembly checkpoint, a molecular protein assembly that inhibits cell division until chromosome alignment is complete. Mutations in several checkpoint genes have been directly linked to cancer, and specific inhibition of checkpoint components has recently been validated as a potent mechanism for selectively killing tumour cells.
Mps1 is a conserved protein kinase whose activity is essential for the spindle assembly checkpoint in all organisms examined, and Mps1 enzymatic activity is subject to regulation by phosphorylation. However, the precise mechanism of Mps1 regulation by phosphorylation is unknown, and this is a direct consequence of our limited biochemical understanding of the checkpoint. The main aim of this study is to develop a focused research programme to investigate checkpoint components at the biochemical level, beginning with the protein kinase Mps1.
I have discovered that bacterial Mps1 can be employed as a model to understand how Mps1 kinase activity is controlled and regulated. To this end, four key objectives will be fulfilled:
1) Molecular dissection of the in vitro mechanism of Mps1 regulation by phosphorylation
2) Analysis of endogenous Mps1 regulation in human cells and Xenopus extracts
3) Regulation of Mps1 activity by exogenous factors present in Xenopus egg extracts
4) Investigation of Mps1 function in human cells by chemical genetics.
A combination of mass spectrometry, mutagenesis and kinase assays will be exploited to examine the first two problems, while biochemical purification and Xenopus extract manipulation will be used to complete the third. Xenopus egg extracts have played a crucial role in advancing our biochemical understanding of the cell cycle, and will be invaluable for these studies. Finally, a cell permeable small molecule kinase inhibitor and a mutant Mps1 target will be exploited to directly assess the physiological regulation of Mps1 in human cells. This study will permit the molecular dissection of requirements for Mps1 activity and create a platform for subsequent studies of other therapeutically relevant protein kinases whose activity is important for diseases such as cancer.