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

Mesio-temporal lobe epilepsy (MTLE) is often accompanied by granule cell dispersion (GCD), a widening of the granule cell layer. The molecular determinants of GCD are poorly understood. Here, we used an animal model to study whether GCD results from an increased dentate neurogenesis associated with an abnormal migration of the newly generated granule cells. Adult mice were given intrahippocampal injections of kainate (KA) known to induce focal epileptic seizures and GCD, comparable to the changes observed in human MTLE. Ipsilateral GCD progressively developed after KA injection and was paralleled by a gradual decrease in the expression of doublecortin, a marker of newly generated granule cells, in the dentate subgranular layer. Staining with Fluoro-Jade B revealed little cell degeneration in the subgranular layer on the KA-injected side. Labeling with bromodeoxyuridine showed an early, transient increase in mitotic activity in the dentate gyrus of the KA-injected hippocampus that gave rise to microglial cells and astrocytes but not to new neurons. Moreover, at later time points, there was a virtually complete cessation of mitotic activity in the injected hippocampus (where GCD continued to develop), but not on the contralateral side (where no GCD was observed). Finally, a significant decrease in reelin mRNA synthesis in the injected hippocampus paralleled the development of GCD, and neutralization of reelin by application of the CR-50 antibody induced GCD. These results show that GCD does not result from increased neurogenesis but reflects a displacement of mature granule cells, most likely caused by a local reelin deficiency.

Mitosis is a highly coordinated process that assures the fidelity of chromosome segregation. Errors in this process result in aneuploidy which can lead to cell death or oncogenesis. In this paper we describe a putative mammalian protein kinase, AIM-1 (Aurora and Ipl1-like midbody-associated protein), related to Drosophila Aurora and Saccharomyces cerevisiae Ipl1, both of which are required for chromosome segregation. AIM-1 message and protein accumulate at G 2 /M phase. The protein localizes at the equator of central spindles during late anaphase and at the midbody during telophase and cytokinesis. Overexpression of kinase-inactive AIM-1 disrupts cleavage furrow formation without affecting nuclear division. Furthermore, cytokinesis frequently fails, resulting in cell polyploidy and subsequent cell death. These results strongly suggest that AIM-1 is required for proper progression of cytokinesis in mammalian cells.

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.

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Disassembly of the nucleolus during mitosis is driven by phosphorylation of nucleolar proteins. RNA processing stops until completion of nucleolar reformation in G(1) phase. Here, we describe the RNA methyltransferase NSUN2, a novel substrate of Aurora-B that contains an NOL1/NOP2/sun domain. NSUN2 was concentrated in the nucleolus during interphase and was distributed in the perichromosome and cytoplasm during mitosis. Aurora-B phosphorylated NSUN2 at Ser139. Nucleolar proteins NPM1/nucleophosmin/B23 and nucleolin/C23 were associated with NSUN2 during interphase. In mitotic cells, association between NPM1 and NSUN2 was inhibited, but NSUN2-S139A was constitutively associated with NPM1. The Aurora inhibitor Hesperadin induced association of NSUN2 with NPM1 even in mitosis, despite the silver staining nucleolar organizer region disassembly. In vitro methylation experiments revealed that the Aurora-B-phosphorylation and the phosphorylation-mimic mutation (S139E) suppressed methyltransferase activities of NSUN2. These results indicate that Aurora-B participates to regulate the assembly of nucleolar RNA-processing machinery and the RNA methyltransferase activity of NSUN2 via phosphorylation at Ser139 during mitosis.