The antiapoptotic DeltaNp73 is degraded in a c-Jun-dependent manner upon genotoxic stress through the antizyme-mediated pathway.

p73, the structural and functional homologue of p53, exists as two major forms: the transactivation-proficient, proapoptotic TAp73 or the transactivation-deficient, antiapoptotic DNp73. Expectedly, expression of both these major forms has to be coordinated precisely to achieve the desired cellular outcome. Genotoxic insults resulting in cell death lead to the stabilization of TAp73, mainly through posttranslational modifications, and the concomitant degradation of DNp73, through poorly understood mechanisms. We have therefore investigated the possible mechanisms of stress-induced DNp73 degradation and show here that c-Jun, the AP-1 family member activated by stress signals and involved in stabilizing TAp73, promotes DNp73 degradation. Genotoxic stress-mediated DNp73 degradation was found to occur in a c-Jun-dependent manner through a ubiquitin-independent but proteasome-dependent mechanism. Absence or down-regulation of c-Jun expression abrogated the reduction of DNp73 levels upon stress insults, whereas overexpression of c-Jun led to its degradation. c-Jun controlled DNp73 degradation through the nonclassical, polyamine-induced antizyme (Az) pathway by regulating the latter's processing during stress response. Consistently, expression of c-Jun or Az, or addition of polyamines, promoted DNp73 degradation, whereas silencing Az expression or inhibiting Az activity in cells exposed to stress reduced c-Jun-dependent DNp73 degradation. Moreover, Az was able to bind to DNp73. These data together demonstrate the existence of a c-Jun-dependent mechanism regulating the abundance of the antiapoptotic DNp73 in response to genotoxic stress.

Downregulation of Aurora-A overrides estrogen-mediated growth and chemoresistance in breast cancer cells.

Estrogen is known to play a causative role in the development of sporadic breast cancers and chemoresistance. However, studies on the mechanism and proteins involved in mediating the oncogenic effects of estrogen are very limited. Recently, Aurora-A, a centrosomal protein kinase, which induces centrosome amplification and genomic instability, has been shown to be upregulated during long-term treatment of rats with estrogen and was implicated in estrogen-induced oncogenesis. Herein, we present results of the studies carried out in short-term in vitro cultures to understand the regulation of Aurora-A by estrogen and the effect of downregulation of Aurora-A on estrogen-induced breast tumorigenesis and chemoresistance. Treatment of breast cancer cells with 10 nM 17beta-estradiol (E(2)) resulted in the upregulation of Aurora-A levels in an estrogen receptor-dependent manner. However, the upregulation by E(2) was not restricted to Aurora-A alone. Following release from the tamoxifen-induced arrest, the appearance of Aurora-A in the presence of estradiol in MCF7 cells coincided with the appearance of other mitotic markers suggesting that the spike in Aurora-A levels is an indirect consequence of estrogen-mediated cell proliferation. Thus, at least in short-term in vitro studies, Aurora-A is not a specific direct target of estrogen. However, downregulation of Aurora-A by RNA interference led to a significant decrease in estrogen-induced, anchorage-dependent, and independent growth of MCF7 cells. Moreover, knockdown of Aurora-A could overcome estrogen-induced decrease in docetaxel sensitivity of MCF7 cells. Cumulatively, we propose that the upregulation of Aurora-A by estrogen in short-term in vitro cultures is an indirect consequence of estrogen-induced cell proliferation. Nevertheless, Aurora-A inhibitors could be exploited to override the effects of estrogen on breast tumorigenesis and chemoresistance.

Aurora-A kinase interacting protein 1 (AURKAIP1) promotes Aurora-A degradation through an alternative ubiquitin-independent pathway.

Mitotic Aurora-A is an oncogene, which undergoes a cell-cycle-dependent regulation of both its synthesis and degradation. Overexpression of Aurora-A leads to aneuploidy and cellular transformation in cultured cells. It has been shown that the cell-cycle-dependent turnover of Aurora-A is mediated by Cdh1 (CDC20 homologue 1) through the anaphase-promoting complex/cyclosome (APC/C)-ubiquitin-proteasome pathway. We have described previously the identification of an Aurora-A kinase interacting protein, AURKAIP1 (formerly described as AIP), which is also involved in the destabilization of Aurora-A through the proteasome-dependent degradation pathway. In an attempt to investigate the mechanism of AURKAIP1-mediated Aurora-A degradation, we report here that AURKAIP1 targets Aurora-A for degradation in a proteasome-dependent but Ub (ubiquitin)-independent manner. AURKAIP1 inhibits polyubiquitination of Aurora-A. A non-interactive AURKAIP1 mutant that cannot destabilize Aurora-A restores ubiquitination of Aurora-A. An A-box mutant of Aurora-A, which cannot be targeted for proteasome-dependent degradation by Cdh1, can still be degraded by AURKAIP1. Inhibition of cellular ubiquitination either by expression of dominant negative Ub mutants or by studies in ts-20 (temperature sensitive-20) CHO (Chinese-hamster ovary) cell line lacking the E1 Ub activating enzyme at the restrictive temperature, cannot abolish AURKAIP1-mediated degradation of Aurora-A. AURKAIP1 specifically decreases the stability of Aurora-A in ts-20 CHO cells at the restrictive temperature, while cyclinB1 and p21 are not affected. This demonstrates that there exists an Ub-independent alternative pathway for Aurora-A degradation and AURKAIP1 promotes Aurora-A degradation through this Ub-independent yet proteasome-dependent pathway.

Aurora-A kinase interacting protein (AIP), a novel negative regulator of human Aurora-A kinase.

Aurora kinases have evolved as a new family of mitotic centrosome- and microtubule-associated kinases that regulate the structure and function of centrosomes and spindle. One of its members, Aurora-A, is a potential oncogene. Overexpression of Aurora-A is also implicated in defective centrosome duplication and segregation, leading to aneuploidy and tumorigenesis in various cancer cell types. However, the regulatory pathways for mammalian Aurora-A are not well understood. Exploiting the lethal phenotype associated with the overexpression of Aurora-A in yeast, we performed a dosage suppressor screen in yeast and report here the identification of a novel negative regulator of Aurora-A, named AIP (Aurora-A kinase Interacting Protein). AIP is a ubiquitously expressed nuclear protein that interacts specifically with human Aurora-A in vivo. Ectopic expression of AIP with Aurora-A in NIH 3T3 and COS cells results in the down-regulation of ectopically expressed Aurora-A protein levels, and this down-regulation is demonstrated to be the result of destabilization of Aurora-A through a proteasome-dependent protein degradation pathway. A noninteracting deletion mutant of AIP does not down-regulate Aurora-A protein, suggesting that the interaction is important for the protein degradation. AIP could therefore be a potential useful target gene for anti-tumor drugs.