Regulation der humanen Aurora-A Kinase und Identifikation potentieller Interaktionspartner

The error-free segregation of duplicated chromosomes during cell
division is essential for the maintenance of an intact genome. This
process is brought about by a highly dynamic bipolar array of
microtubules, the mitotic spindle. Prominent amongst the regulators
of the mitotic spindle is the serine/threonine-specific protein
kinase Aurora-A. However, only a few interaction partners and
physiological substrates of Aurora-A are know to date and the
function of this important kinase is only beginning to emerge. To
gain further insight in the cellular function of Aurora-A I set out
studies to characterize Aurora-A and to identify new interaction
partners of Aurora-A. To characterize the cellular function of
Aurora-A the siRNA phenotype of Aurora-A was analysed. This showed
that Aurora-A depletion in human HeLa cells led to apoptosis and
spindle defects. To identify new Aurora-A interacting proteins a
polyclonal antibody against Aurora-A was generated and used in
co-immunoprecipitations from cell extracts. In addition
Yeast-Two-Hybrid Screening and fishing experiments with recombinant
proteins were performed. These studies provided four new putative
Aurora-A interacting partners (KIAA1007, KIAA1741, TPX2 and a
candidate from the Yeast-Two-Hybrid Screen). The interaction of
these proteins with Aurora-A was verified using different
biochemical methods. KIAA1741 and TPX2 were analysed in more
detail. A polyclonal antibody against KIAA1741 was generated and
KIAA1741 was shown to localize to actin rich structures within the
cell. KIAA1741 interacted with recombinant Aurora-A showing a
higher affinity for the catalytically active kinase in mitosis.
Depletion of KIAA1741 by siRNA induced a G1 arrest. Interestingly
KIAA1741 was a good in vitro substrate of Aurora-A. Most
importantly this study revealed that Aurora-A binds to TPX2, a
known component of the spindle apparatus. Binding studies
demonstrated that the first 43 amino acids of TPX2 were sufficient
and necessary for the interaction with the C-terminal catalytic
domain of Aurora A. Although kinase activity was not required for
this interaction, TPX2 was readily phosphorylated by Aurora-A on
serine residues. Upon siRNA-mediated elimination of TPX2 from
cells, the association of Aurora-A with the spindle microtubules
was abolished. Furthermore these experiments showed that TPX2 was
essential for the formation of a bipolar spindle and spindle pole
integrity. Moreover I could demonstrate that TPX2 activates the
Aurora-A kinase activity in a microtubule dependant manner,
indicating that TPX2 is a new regulator of Aurora-A.