Aurora kinase A is a member of a new family of serine/threonine kinases that includes Drosophila melanogaster Aurora and Saccharomyces cerevisiae Ipl1 kinase, both of which are essential for controlling normal chromosome segregation and centrosome functions [1][2][3] . Aurora kinase A has been implicated in regulating centrosome function, spindle assembly, spindle maintenance and mitotic commitment in cells [4][5][6][7] . AURKA, encoding aurora kinase A, is a putative oncogene that is amplified and overexpressed in many human cancers [8][9][10][11][12][13] . The molecular targets of aurora kinase A have not been well characterized. We previously reported that phosphorylationmediated feedback between aurora kinase A and protein phosphatase 1 operates through mitosis and that disruption of this interaction results in defects in chromosome segregation 14 .Overexpression of aurora kinase A 8 and loss of wild-type p53 function induce similar phenotypes of centrosome amplification and aneuploidy in cells 15,16 . These observations suggest that gain of aurora kinase A function and loss of wild-type p53 function may be interdependent in common pathways. The finding that human tumors with elevated expression of aurora kinase A have wild-type TRP53 (encoding p53) also suggests that gain of aurora kinase A function may cause loss of wild-type p53 function, contributing to malignant transformation. p53 induces growth arrest or apoptosis in cells exposed to stress and is frequently mutated or deleted in human cancers. Expression of p53 is controlled by Mdm2, which promotes ubiquitination by E3 ubiquitin ligase activity and degradation of p53 by the cytoplasmic 26S proteasome 17 . Stability and activity of p53 are also regulated by post-translational modifications 18-23 including phosphorylation, acetylation, glycosylation and attachment of a small ubiquitin-related modifier protein. Phosphorylation at multiple sites is the predominant mechanism known to stabilize and abrogate Mdm2-mediated ubiquitination and activates p53. In contrast, phosphorylation of the core domain at Thr155 by the COP9 signalosome has been reported to target p53 for degradation 24 . The present study investigated whether phosphorylation by aurora kinase A also regulates p53 activity. RESULTS Aurora kinase A phosphorylates and interacts with p53We first investigated the ability of aurora kinase A to phosphorylate p53 in an in vitro kinase assay. We incubated bacterially expressed glutathione S-transferase (GST) and a GST-p53 fusion protein with aurora kinase A immunoprecipitated from mitotic HeLa cells and γ 32 P ATP. The aurora kinase A immunocomplex clearly phosphorylated GST-p53 (Fig. 1a). To confirm the specificity of aurora kinase A in phosphorylating p53, we used immunoprecipitated wild-type and kinase-inactive aurora kinase A (K162R) in an in vitro kinase assay with GST-p53. Wild-type aurora kinase A phosphorylated p53 but the kinase-inactive mutant did not (Fig. 1b), confirming that aurora A R T I C L E S

The function of Aurora-C kinase, a member of the Aurora kinase family identified in mammals, is currently unknown. We present evidence that Aurora-C, like Aurora-B kinase, is a chromosomal passenger protein localizing first to centromeres and then to the midzone of mitotic cells. Aurora-C transcript is expressed at a moderate level albeit about an order of magnitude lower than Aurora-B transcript in diploid human fibroblasts. The level of Aurora-C transcript is elevated in several human cancer cell types. Aurora-C and Aurora-B mRNA and protein expressions are maximally elevated during the G2/M phase but their expression profiles in synchronized cells reveal differential temporal regulation through the cell cycle with Aurora-C level peaking after that of Aurora-B during the later part of the M phase. Aurora-C, like Aurora-B, interacts with the inner centromere protein (INCENP) at the carboxyl terminal end spanning the conserved IN box domain. Competition binding assays and transfection experiments revealed that, compared with Aurora-C, Aurora-B has preferential binding affinity to INCENP and co-expression of the two in vivo interferes with INCENP binding, localization, and stability of these proteins. A kinase-dead mutant of Aurora-C had a dominant negative effect inducing multinucleation in a dose-dependent manner. siRNA mediated silencing of Aurora-C and Aurora-B also gave rise to multinucleated cells with the two kinases silenced at the same time displaying an additive effect. Finally, Aurora-C could rescue the Aurora-B silenced multinucleation phenotype, demonstrating that Aurora-C kinase function overlaps with and complements Aurora-B kinase function in mitosis.

Summary Elevated Aurora kinase-A expression is correlated with abrogation of DNA damage induced apoptotic response and mitotic spindle assembly checkpoint (SAC) override in human tumor cells. We report that Aurora-A phosphorylation of p73 at serine235 abrogates its transactivation function and causes cytoplasmic sequestration in a complex with the chaperon protein mortalin. Aurora-A phosphorylated p73 also facilitates inactivation of SAC through dissociation of the MAD2-CDC20 complex in cells undergoing mitosis. Cells expressing phosphor-mimetic mutant (S235D) of p73 manifest altered growth properties, resistance to cisplatin induced apoptosis, as well as premature dissociation of the MAD2-CDC20 complex, and accelerated mitotic exit with SAC override in the presence of spindle damage. Elevated cytoplasmic p73 in Aurora-A overexpressing primary human tumors corroborates the experimental findings.

Aurora-A/BTAK/STK15 localizes to the centrosome in the G2-M phase, and its kinase activity regulates the G2to M transition of the cell cycle. Previous studies have shown that the BRCA1 breast cancer tumor suppressor also localizes to the centrosome and that BRCA1 inactivation results in loss of the G2-M checkpoint. We demonstrate here that Aurora-A physically binds to and phosphorylates BRCA1. Biochemical analysis showed that BRCA1 amino acids 1314–1863 binds to Aurora-A. Site-directed mutagenesis indicated that Ser308of BRCA1 is phosphorylated by Aurora-Ain vitro. Anti-phospho-specific antibodies against Ser308of BRCA1 demonstrated that Ser308is phosphorylatedin vivo. Phosphorylation of Ser308increased in the early M phase when Aurora-A activity also increases; these effects could be abolished by ionizing radiation. Consistent with these observations, acute loss of Aurora-A by small interfering RNA resulted in reduced phosphorylation of BRCA1 Ser308, and transient infection of adenovirus Aurora-A increased Ser308phosphorylation. Mutation of a single phosphorylation site of BRCA1 (S308N), when expressed in BRCA1-deficient mouse embryo fibroblasts, decreased the number of cells in the M phase to a degree similar to that with wild type BRCA1-mediated G2arrest induced by DNA damage. We propose that BRCA1 phosphorylation by Aurora-A plays a role in G2to M transition of cell cycle.

Microtubule nucleation and formation from the kinetochore/ chromatin have been proposed to contribute to bipolar spindle assembly facilitating equal segregation of chromosomes in mitosis. Although two independent pathways involving the small Ran GTPase-TPX2 proteins and the chromosomal passenger complex proteins have been implicated in the formation of microtubules from the kinetochore/chromatin, detailed molecular mechanisms integrating the pathways and regulating the process have not been well elucidated. This study demonstrates that Aurora kinase-A plays a central role in the kinetochore/chromatin associated microtubule assembly in human cells by integrating the two pathways regulating the process. Silencing by siRNA and overexpression of a kinase inactive mutant revealed involvement of Aurora-A at two critical steps. These include formation of γ-tubulin foci in the vicinity of kinetochore/chromatin to create microtubule nucleation sites as well as INCENP and TPX2 mediated activation of Aurora-A facilitating formation and stabilization of microtubules. The findings provide the first evidence of Aurora-A, in association with INCENP and TPX2, being a key regulator of kinetochore/ chromatin associated microtubule formation in human cells.