Aurora-A Kinase Maintains the Fidelity of Early and Late Mitotic Events in HeLa Cells

Abstract

Aurora-A, a member of the Aurora/Ipl1-related kinase family, is overexpressed in various types of cancer and considered to play critical roles in tumorigenesis. To better understand the pathological effect of Aurora-A activation, it is first necessary to elucidate the physiological functions of Aurora-A. Here, we have investigated the roles of Aurora-A in mitotic progression with the small interfering RNA, antibody microinjection, and time lapse microscopy using human cells. We demonstrated that suppression of Aurora-A by small interfering RNA caused multiple events to fail in mitosis, such as incorrect separation of centriole pairs, misalignment of chromosomes on the metaphase plate, and incomplete cytokinesis. Antibody microinjection of Aurora-A into late G 2 cells induced dose-dependent failure in separation of centriole pairs at prophase, indicating that Aurora-A is essential for proper separation of centriole pairs. When we injected anti-Aurora-A antibodies into prometaphase cells that had separated their centriole pairs, chromosomes were severely misaligned on the metaphase plate, indicating that Aurora-A is required for proper movement of chromosomes on the metaphase plate. Furthermore, inhibition of Aurora-A at metaphase by microinjected antibodies prevented cells from completing cytokinesis, suggesting that Aurora-A also has important functions in late mitosis. These results strongly suggest that Aurora-A is essential for many crucial events during mitosis and that the phosphorylation of a series of substrates by Aurora-A at different stages of mitosis may promote diverse critical events in mitosis to maintain chromosome integrity in human cells.

BimEL is phosphorylated at mitosis by Aurora A and targeted for degradation by βTrCP1

“…The siRNA oligonucleotide sequence targeting bTRCP1/2 has been described previously 54 and corresponds to nt 515-535 of human bTRCP1 and nt 262-282 of human bTRCP2. The following siRNA sequences were used to knock down Aurora A: 55 5 0 -725 AUGCCC UGUCUUACUGUCA 743 -3 0 and 5 0 -155 AUUCUUCCCAGCGCGUUCC 173 -3 0 . To knock down PP2A catalytic subunit, we used the following siRNA sequences Hs_PPP2CA_5 5 0 -ATGGAACTTGACGATACTCTA-3 0 and Hs_PPP2CA_6 5 0 -CAAACAATCATTGGAGCTTAA-3 0 .…”

Phosphorylation of human enhancer of filamentation (HEF1) on serine 369 induces its proteasomal degradation

“…The Aurora-A and -B small interfering RNA (siRNA) sequences used have been described previously and are as follows : 5'-AUGCCCUGUCUUACUGUCA-3' (Aurora-A) and 5'-GGUGAUGGAGAAUAGCAGU-3' (Aurora-B) [31,33]. Briefly, 10 or 20 pmol of siRNA and 20 µl of Interferin (Ozyme) were added to 200 l of Opti-MEM (Invitrogen), mixed gently, and then incubated for 10 minutes at room temperature.…”

Aurora A overexpression induces cellular senescence in mammary gland hyperplastic tumors developed in p53-deficient mice

“…Dysfunction of some mitotic kinases leads to chromosome rearrangements and aneuploidy, which are hallmarks of many human cancers, indicating that mitotic kinases are important for maintaining genomic integrity (Nigg, 2001). Aurora A serine/threonine mitotic kinase has crucial roles in various mitotic events such as centrosome maturation and separation, mitotic entry, bipolar spindle assembly, chromosome alignment and cytokinesis (Meraldi et al, 2002;Hirota et al, 2003;Kunitoku et al, 2003;Marumoto et al, 2003Marumoto et al, , 2005Giet et al, 2005).…”