Identifying new shared substrates of Aurora kinases at the mitotic apparatus

Abstract

Aurora A and B are the major kinases that control key events in mitosis, such as centrosome function, spindle assembly, chromosome segregation and cytokinesis, through phosphorylation of multiple proteins. These kinases share identical consensus target motifs, so the substrate specificity is determined by distinctive sub-cellular localization of the Auroras. Many proteins have been identified as targets of either Aurora A, or Aurora B, or both kinases by mass spectrometry studies. However, only a few of the identified phosphorylation sites in these targets have a characterized function in vivo. Therefore, the molecular mechanisms underlying the regulation of certain mitotic events by Aurora kinases remain unclear. The objective of my work was to develop a tool for identifying new substrates of both Aurora kinases. More specifically, I aimed to identify the molecular targets of Aurora A at the kinetochores, and determine how Aurora A contributes to the error correction near spindle poles. I first demonstrated that the outer kinetochore protein HEC1/Ndc80, phosphorylated by Aurora B at kinetochores, can also be phosphorylated by Aurora A close to the centrosomes (Chapter 2). My finding showed that Aurora kinases can share substrates in the cells and revealed the mechanism by which Aurora A contributes to the error-correction near spindle poles. To identify and characterise novel substrates of Aurora kinases, I developed a bioinformatic approach in collaboration with the Centre Bioinformatician, Alastair Kerr. This bioinformatic method uses the Auroras’ shared consensus motifs combined with several parameters that control the substrate specificity of Aurora kinases. I tested the phosphorylation of the chosen candidates in vitro using radiolabelled kinase assays. In my study, five proteins were validated - SPICE1, TTLL4, AHCTF1, CLASP2 and an uncharacterized protein KIAA1468 - as in vitro substrates of Aurora A and Aurora B kinases (Chapter 3). I then focussed on the Aurora kinases-dependent regulation of spindle and centriole-associated protein, SPICE1, in cells (Chapter 4). Using either site-directed mutagenesis of SPICE1 or inhibition of Aurora kinases with small molecule inhibitors, I found that the predicted phosphorylation of the SPICE1 C terminus had the function in cells of directing the SPICE1 localization on the spindle MTs. My results demonstrate the high accuracy of this genome-wide bioinformatics approach. By complementing mass spectrometry studies, here lies a potential for the identification of other unknown substrates, which is important for the general understanding of how Aurora kinases regulate the mitotic apparatus.