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.

Level thesis:degree_level

thesis.doctoral

Grantor dc:publisher

Ludwig-Maximilians-Universität

Year

2004

Author dc:creator

• Kufer, Thomas

Repository record source_url

https://edoc.ub.uni-muenchen.de/1960/

OAI identifier oai:identifier

oai:edoc.ub.uni-muenchen.de:1960