Exploring the regulation and function of human Lats1 and Aurora A kinases in mitosis

Mitosis is the process by which sister chromatids are equally
segregated into two daughter cells. Tight control in various events
during mitotic progression is essential for maintaining chromosome
stability. Mitotic kinases including Cyclin dependent kinase 1
(Cdk1) and Aurora family are required for regulating proper mitotic
progression by phosphorylating mitotic substrates thereby,
controlling their activities, localization or abundance. On the
other hand, these mitotic kinases are modulated by de-novo
synthesis, activators, phosphorylation and ubiquitin-dependent
proteolysis. A thorough understanding of the function and
regulation of mitotic kinases could further our knowledge on
mitotic progression. In the first part of the thesis, we
investigated the expression, localization and regulation of human
Lats1 kinase, which is a close homologue of the yeast Dbf2 kinase
family involved in the mitotic exit network (MEN). Despite the fact
that Lats1 has been suggested to be a spindle protein that binds
and inactivates Cdk1, we found that Lats1 is mainly cytoplasmic
throughout the cell cycle by immunofluorescence microscopy. Both
yeast two-hybrid and coimmunoprecipitation showed no significant
interaction between Lats1 and Cdk1. Although Lats1 was highly
phosphorylated during mitosis, no detectable kinase activity was
observed. However, we identified Ste20 like kinase MST2 as the
upstream regulator of human Lats1. Phosphorylation of Lats1 by Mst2
resulted in the activation of Lats1 kinase activity both in vivo
and in vitro. This kinase-substrate relation was proven to be
specific, as another distant Mst2 homolog, Mst4, did not possess
this ability. Subsequent mass-spectrometry-based phosphosites
analysis revealed that Mst2 phosphorylates Lats1 on more than five
residues. Alanine mutations on Lats1T1079 and S909 impaired Lats1
kinase activity. Thus, we could not confirm the suggested role of
Lat1 in mitosis. Instead, we show that similar to its Drosophila
ortholog, Lats1 is involved in the Mst2 signaling pathway and might
control developmentally regulated cell proliferation and apoptosis
in mammals. In the second part of this thesis, we characterized
hBora, a novel Aurora A interactor originally found in Drosophila.
We show that hBora is upregulated and phosphorylated during
mitosis. siRNA-mediated knockdown of hBora led to spindle formation
defects and aneuploidy. hBora overexpression caused monoastral
spindle formation and mislocalization not only of Aurora A but also
Plk1. Further investigations showed that Cdk1 phosphorylation on
hBoraSer252 leads to Plk1 binding and this may promote the
SCF-mediated proteolysis of hBora. Indeed, Plk1 depletion led to an
increase in hBora levels. Interestingly, the co-depletion of both
hBora and Plk1 (to lower hBora levels in Plk1 depleted cells)
rescued the localization of Aurora A to the centrosomes and bipolar
spindle formation. Thus, we propose that hBora is a functional link
between Plk1 and Aurora A and that by modulating the proteolysis of
hBora, Plk1 could regulate Aurora A localization and activity. At
the end, we also investigated the function of Aurora A and could
show that Aurora A is required for centriole cohesion and
centrosome separation.