Functional Analysis of Aurora-A kinase – AURORA

The serine/theronine kinase Aurora-A is a key player in mitosis. Although discovered in 1998 its functions are not fully understood yet. Inhibition of its activity by RNA interference leads to a defect in centrosome maturation and consequently the formation of abnormal spindles during mitosis. This result was obtained because it is the first event in which Aurora-A kinase activity is required during cell cycle. Whether the kinase is necessary later in mitosis : during metaphase to anaphase progression for instance, or during cytokinesis or for exit from mitosis is not known because it is difficult to test. One possible approach would be to specifically target the kinase with an inhibitor. But to date there is not any specific inhibitor of Aurora-A available. Thus it is not possible to analyse the function of the kinase in short windows of time during cell cycle progression. To overcome this problem we have been working to design a genetically modified Aurora-A kinase that behaves just like the wild type kinase but that is sensitive to an chemical inhibitor that has no target in normal cell and that is easy to manipulate because it rapidly enters the cells. We have obtained such a genetically modified Aurora-A kinase sensitive to a specific inhibitor (NaPP1) that we call as-AurA. We have validated the use of as-AurA kinase together with its inhibitor NaPP1 using RNAi/rescue. We found that Aurora A is indeed required in late stages of mitosis. Because the RNAi/rescue approach is too complicated we decided to find a way to design cells, which would encode only for as-AurA kinase. An Aurora-A PM/cKO mouse is currently under construction in Strasbourg/ As soon as we obtain the mouse we will be able to answer the following questions 1 ' Is Aurora-A kinase activity required early on during oocyte maturation or only at meiosis 1' This is a crucial question that has remained without unambiguous answer since 1998. Conflicting reports keep being published on this matter. Incubation of immature oocytes from Aurora-A PM/cKO female will give a straight answer. 2 ' Is Aurora-A activity required during mitosis after metaphase' Is it involved in chromosome segregation' Is it required for cytokinesis' for abscission' etc'. With this Aurora-A PM/cKO mouse we will have access to MEF cells containing only as-AurA sensitive to NaPP1. We plan to use the inhibitor NaPP1 coupled with video-microscopy. 3 ' Is a very specific inhibition of Aurora-A a good strategy to treat cancers' This is crucial for cancer research. We will have accessed for the first time to an animal in which we could analyse the effect of an AurA inhibition. We will also induce tumour formation and evaluate the effect of a very specific Aurora-A inhibition on tumour regression. 4 ' Is Aurora-A a tumour suppressor in mammalian' Inducible Aurora-A KO in tissues in which asymmetric divisions occur at high rate will allow us to test this. 5 ' How to purify and identify Aurora-A substrates' This last project is conducted in collaboration with chemists in Rennes.