Aurora-A and an Interacting Activator, the LIM Protein Ajuba, Are Required for Mitotic Commitment in Human Cells

Abstract

Aurora family kinases contribute to regulation of mitosis. Using RNA interference in synchronized HeLa cells, we now show that Aurora-A is required for mitotic entry. We found that initial activation of Aurora-A in late G2 phase of the cell cycle is essential for recruitment of the cyclin B1-Cdk1 complex to centrosomes, where it becomes activated and commits cells to mitosis. A two-hybrid screen identified the LIM protein Ajuba as an Aurora-A binding protein. Ajuba and Aurora-A interact in mitotic cells and become phosphorylated as they do so. In vitro analyses revealed that Ajuba induces the autophosphorylation and consequent activation of Aurora-A. Depletion of Ajuba prevented activation of Aurora-A at centrosomes in late G2 phase and inhibited mitotic entry. Overall, our data suggest that Ajuba is an essential activator of Aurora-A in mitotic commitment.

Overexpression of aurora kinase A in mouse mammary epithelium induces genetic instability preceding mammary tumor formation

“…Aurora-A is localized at the centrosome during interphase, translocated to the mitotic spindle in early mitotic phase and degraded after metaphase transition. It has been demonstrated that activation of Aurora-A is required for mitotic entry, centrosome maturation and separation and G2 to M transition (Marumoto et al, 2002;Meraldi et al, 2002;Anand et al, 2003;Hirota et al, 2003;Giet et al, 2005). Overexpression of Aurora-A also results in defective spindle assembly checkpoint, allowing cells with abnormal chromosomal separation to enter anaphase, leading to aneuploidy (Zhou et al, 1998;Stenoien et al, 2003).…”

Phosphorylation of the cytokinesis regulator ECT2 at G2/M phase stimulates association of the mitotic kinase Plk1 and accumulation of GTP-bound RhoA

“…Mitotic kinases, such as Cdk1, polo-like kinase 1 (Plk1), Aurora-A, and Aurora-B, are known to play a critical role in mitotic events including centrosome separation, chromosome condensation, nuclear envelope breakdown, and microtubule reorganization (Nigg et al, 1996;Nigg, 1998Nigg, , 2001Severson et al, 2000;Hirota et al, 2003). The serine/threonine protein kinase Plk1 contributes to normal mitotic phase progression, and localizes at mitotic spindles in metaphase and then at the midbody in cytokinesis (Nigg et al, 1996;Yuan et al, 2002).…”

AIP regulates stability of Aurora-A at early mitotic phase coordinately with GSK-3β

“…More than 96% of AIP knockdown cells were in prophase to metaphase, whereas 72% of control cells were in these mitotic phases (Figure 3b), suggesting that knockdown of AIP results in arrest in early mitotic phase. To further characterize the phenotype of AIP knockdown cells, we followed individual mitotic cells using time-lapse microscopy in HeLa cells stably expressing histone H2B-GFP (HeLa/H2B-GFP cells) (Hirota et al, 2003). A delay of exit from metaphase was observed upon knockdown of AIP (Figure 3c).…”

“…HeLa cells expressing histone H2B-GFP (HeLa/H2B-GFP), and polyclonal anti-Aurora-A antibody were generated as described (Hirota et al, 2003). A synthesized peptide of the middle portion (residues 103-120) or C-terminal fragment (residues 183-199) of AIP was used to generate anti-AIP antibody in a rabbit.…”

“…The action of TPX2 on Aurora-A is mediated by small GTPase Ran that facilitates microtubule polymerization and bipolar spindle assembly (Tsai et al, 2003). Regulation of the stability and activity of Aurora-A is important for mitotic progression, because reduction of Aurora-A in the cells causes delay in mitotic entry (Hirota et al, 2003) and overexpression of Aurora-A leads to abnormal centrosome amplification, chromosomal instability and transformation (Bischoff et al, 1998). Aurora-A is located in the chromosome 20q13 region, a region that is frequently amplified in breast cancers and in diverse cancer cell lines.…”