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

MDMX, an MDM2-related protein, has emerged as yet another essential negative regulator of p53 tumor suppressor, since loss of MDMX expression results in p53-dependent embryonic lethality in mice. However, it remains unknown why neither homologue can compensate for the loss of the other. In addition, results of biochemical studies have suggested that MDMX inhibits MDM2-mediated p53 degradation, thus contradicting its role as defined in gene knockout experiments. Using cells deficient in either MDM2 or MDMX, we demonstrated that these two p53 inhibitors are in fact functionally dependent on each other. In the absence of MDMX, MDM2 is largely ineffective in down-regulating p53 because of its extremely short half-life. MDMX renders MDM2 protein sufficiently stable to function at its full potential for p53 degradation. On the other hand, MDMX, which is a cytoplasmic protein, depends on MDM2 to redistribute into the nucleus and be able to inactivate p53. We also showed that MDMX, when exceedingly overexpressed, inhibits MDM2-mediated p53 degradation by competing with MDM2 for p53 binding. Our findings therefore provide a molecular basis for the nonoverlapping activities of these two p53 inhibitors previously revealed in genetic studies.The tumor suppressor gene p53 encodes a transcription factor that is activated in response to various forms of stress, leading to the induction of a number of genes whose products mediate either cell cycle arrest or apoptosis (1). Under most physiological conditions, p53 activity is tightly controlled, primarily through the ability of MDM2 to target p53 for degradation, which ensures cell survival. Current model of p53 activation suggests that diverse stress signals converge on a single regulatory node, namely the p53-MDM2 module, and interfere with the ability of MDM2 to target p53 for degradation (2). Analogous to MDM2, MDMX ablation is also associated with p53-dependent embryonic death in mice, placing MDMX in the category of essential p53 negative regulators (3). In contrast to MDM2, however, MDMX lacks ubiquitin E3 ligase activity and is unable to target p53 for ubiquitin-proteasome-dependent proteolysis (4). Moreover, MDMX was reported to inhibit MDM2-mediated p53 degradation (4 -6), contradicting the role of MDMX as defined by the genetic study. To resolve these conflicting results and gain better understanding of why neither gene product can compensate for the loss of the other, we generated MDMX-deficient cells using small interference RNA (siRNA) 1 and carried out biochemical analysis of MDM2 in these cells. In conjunction with the use of MEFs derived from either single or double knock-out mice, our loss-of-function approach allowed us to obtain compelling evidence at the molecular level to highlight mutual dependence of MDM2 and MDMX in their functional inhibition of p53 and provide support for the findings obtained in genetic studies. /MDM2Ϫ/Ϫ MEFs (Dr. Carl Maki, Harvard School of Public Health), were maintained in minimal essential medium supplemented with 10% fetal bovin...

Although genetic studies have demonstrated that MDMX is essential to maintain p53 activity at low levels in non-stressed cells, it is unknown whether MDMX regulates p53 activation by DNA damage. We show here that DNA damage-induced p53 induction is associated with rapid down-regulation of the MDMX protein. Significantly, interference with MDMX down-regulation results in the suppression of p53 activation by genotoxic stress. We also demonstrate that DNA damage-induced MDMX reduction is mediated by MDM2, which targets MDMX for proteasomal degradation by a distinct mechanism that permits preferential MDMX degradation and therefore ensures optimal p53 activation.

The c-Abl nonreceptor tyrosine kinase is activated in cells exposed to ionizing radiation (IR) 1 and certain other DNAdamaging agents (1-4). IR induces DNA double-strand breaks (5) and thereby activates the DNA-dependent protein kinase (DNA-PK) (6 -8). Recent work has shown that DNA-PK phosphorylates and activates c-Abl (9). Other studies have demonstrated that c-Abl interacts with the ataxia telangiectasia mutated (ATM) gene product and that ATM may activate c-Abl in the response to genotoxic stress (10, 11). Whereas cells deficient in DNA-PK or ATM are hypersensitive to killing by IR (12, 13), c-Abl-deficient cells are resistant to IR-induced apoptosis (14). Activation of c-Abl by genotoxic stress is associated with interaction of c-Abl with the p53 tumor suppressor in the G 1 arrest response (15,16). Other signals dependent on c-Abl activation include induction of the stress-activated protein kinase and p38 mitogen-activated protein kinase by genotoxic agents (1,2,17). The findings that c-Abl contributes to the regulation of p53 and certain stress-induced kinases associated with apoptosis have provided support for the activation of c-Abl as a pro-apoptotic signal (14). In this context, expression of c-Abl is associated with G 1 phase growth arrest and induction of apoptosis (14,18,19).Recombination plays a fundamental role in the repair of DNA damage. In Escherichia coli, the RecA protein mediates repair of double-strand breaks by initiating pairing and strand exchange between homologous DNAs (20). Identification of structural homologs of RecA in yeast, Xenopus laevis, mouse, and human cells has supported conservation of similar repair functions throughout evolution (21-25). ScRad51, the RecA homolog in Saccharomyces cerevisiae, is required for DNA damage-induced mitotic recombination (21). ScRad51 converts DNA double-strand breaks to recombinational intermediates, and rad51 mutants accumulate these breaks during meiosis (21). The finding that human Rad51 (HsRad51) promotes homologous pairing and strand exchange reactions in vitro has suggested that Rad51 may also play a role in recombinational repair in man (26). Whereas yeast deficient in Rad51 are viable (21), targeted disruption of the rad51 gene in mice results in an embryonic lethal phenotype (27,28). These findings in rad51 Ϫ/Ϫ mice have suggested that mammalian Rad51 has an essential role in cell proliferation and/or maintenance of genomic stability.The present studies demonstrate that c-Abl associates with Rad51. We show that c-Abl phosphorylates Rad51 on Tyr-54 in vitro and in irradiated cells. Importantly, phosphorylation of Rad51 by c-Abl inhibits Rad51 function in DNA strand exchange assays. MATERIALS AND METHODSCell Culture-U-937 cells, HeLa cells, 293 embryonal kidney cells, and mouse embryo fibroblasts (Abl Ϫ/Ϫ , Abl ϩ ) (29) were grown as described (1). Irradiation was performed using a Gammacell 1000 (Atomic Energy of Canada) with a 137 Cs source emitting at a fixed dose of 0.21 gray min Ϫ1 as determined by dosimetry. Immunoprecipitations ...