Aurora A: Working from dawn to dusk in mitosis

The kinase Aurora A (AurA) has been a focus of attention in basic and applied research since its identification in 1995, in large part because misregulation of its expression and activity has been linked to tumorigenesis and cancer. This has fueled the search for specific inhibitors, leading to the identification of compounds that are currently being tested in clinical trials for their potential antitumor activity. AurA is one of the 3 members of the Serine/Threonine Aurora kinase family in vertebrates. Both AurA and Aurora B are cell cycle-regulated kinases with a peak of protein expression and kinase activity during G 2 /M pointing to major roles during cell division. Although each kinase has a characteristic pattern of localization throughout mitosis and specific subsets of activators and regulators, a major issue has been to clearly determine the specificity toward potential substrates and the level of functional overlap. Characteristically, AurA accumulates at the centrosome throughout mitosis and also localizes along spindle microtubules (MTs). AurA was shown to be essential for bipolar spindle assembly, in large part by promoting MT assembly and organization. The mechanism at play involves the phosphorylation of MT-related proteins, including some involved in MT nucleation, like NEDD1, and others in MT assembly or stabilization, like TACC3, which works in concert with the MT polymerase ch-TOG/XMAP215. The essential role of AurA activity in the assembly of the metaphase spindle had precluded so far functional studies addressing its potential role post-metaphase. However, some data suggested that its role could well extend to the last mitotic phases. Indeed, AurA is only degraded late at the end of mitosis. Moreover, its degradation was found to be necessary for MTs to return to the interphase state. Indirectly, this suggested that AurA activity could be required to prevent MTs to return to interphase prematurely after metaphase, when the main mitotic kinase cyclinB/Cdk1 is switched off. It was therefore tempting to speculate that AurA could have a function after metaphase, potentially contributing to the control of MT dynamics and organization throughout mitosis. This has turned out to be the case. Taking advantage of the selective inhibitor MLN8237, we specifically targeted AurA kinase activity at the metaphase-to-anaphase transition and followed the consequences on chromosome segregation and MT organization by live imaging. The transition from metaphase to anaphase entails a radical change of the phosphorylation landscape and the reorganization of the MT network to support the efficient separation of the sister chromatids to the prospective daughter cells. This involves the fast shortening of the K-fibers that pull the sister chromatids toward the opposite spindle poles and the assembly of a novel MT based machinery, the central spindle. This structure consists of 2 antiparallel arrays of interdigitating MTs that assemble between the segregating chromosomes. By MT sliding, the central spindle contributes to the net separation of the chromosomes, thereby ensuring that the daughter cells receive an equal genomic content. The inhibition of AurA in anaphase impaired the assembly of a robust central spindle, lowering the density of MTs in this area and preventing their neat antiparallel organization. Interestingly this phenotype had some reminiscences with the pre-metaphase phenotype resulting from altering AurA levels or activity, in that both are consistent with MT stability and organization defects. For this reason, and in the absence of an unbiased assay to identify the AurA substrate(s) involved, we decided to focus on one of them, TACC3, which has been well characterized for its role promoting MT assembly in early mitosis, but whose function during anaphase had not been previously addressed. Consistent with previous reports, we found that TACC3-silenced cells did enter anaphase. However, we observed central spindle defects strikingly similar to those found in AurA-inhibited cells. These results implicated TACC3 in central spindle assembly for the first time. We then examined whether its phosphorylation by AurA was required for this novel function, as it is in the early phases of mitosis. Indeed, silencing and rescue experiments using different phosphorylation variants of TACC3 showed that the non-phosphorylatable version of TACC3 could not rescue the defects observed in TACC3 silenced cells. Moreover, they phenocopied the inhibition of AurA activity. Overall our results have provided novel insights into the mechanism involved in central spindle assembly by identifying 2 novel important players: the kinase AurA and its substrate TACC3. The role of AurA during anaphase has also recently been addressed in an