Identifying new shared substrates of Aurora kinases at the mitotic apparatus
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Date
09/07/2018Item status
Restricted AccessEmbargo end date
09/07/2019Author
Deretic, Jovana
Metadata
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.