Aurora-A kinase maintains the fidelity of early and late mitotic events in HeLa cells

Aurora-A, a member of the Aurora/Ipl1-related kinase family, is overexpressed in various types of cancer and considered to play critical roles in tumorigenesis. To better understand the pathological effect of Aurora-A activation, it is first necessary to elucidate the physiological functions of Aurora-A. Here, we have investigated the roles of Aurora-A in mitotic progression with the small interfering RNA, antibody microinjection, and time lapse microscopy using human cells. We demonstrated that suppression of Aurora-A by small interfering RNA caused multiple events to fail in mitosis, such as incorrect separation of centriole pairs, misalignment of chromosomes on the metaphase plate, and incomplete cytokinesis. Antibody microinjection of Aurora-A into late G2 cells induced dose-dependent failure in separation of centriole pairs at prophase, indicating that Aurora-A is essential for proper separation of centriole pairs. When we injected anti-Aurora-A antibodies into prometaphase cells that had separated their centriole pairs, chromosomes were severely misaligned on the metaphase plate, indicating that Aurora-A is required for proper movement of chromosomes on the metaphase plate. Furthermore, inhibition of Aurora-A at metaphase by microinjected antibodies prevented cells from completing cytokinesis, suggesting that Aurora-A also has important functions in late mitosis. These results strongly suggest that Aurora-A is essential for many crucial events during mitosis and that the phosphorylation of a series of substrates by Aurora-A at different stages of mitosis may promote diverse critical events in mitosis to maintain chromosome integrity in human cells.     

Functional interaction of Aurora-A and PP2A during mitosis

Entry into mitosis is a highly regulated process, promoted by the activated Cyclin B1/Cdk1 complex. Activation of this complex is controlled, in part, by the protein kinase Aurora-A, which is a member of a multigenic serine/threonine kinase family. In normal cells, Aurora-A activity is regulated, at least in part, by degradation through the APC-ubiquitin-proteasome pathway. It has recently been proposed that, in Xenopus, Aurora-A degradation can be inhibited by phosphorylation. It would thus be expected that a phosphatase activity would release this blockade at the end of mitosis. Here, we have shown that the protein phosphatase PP2A and Aurora-A are colocalized at the cell poles during mitosis in human cells and interact within the same complex. Using the PP2A inhibitor okadaic acid and an RNAi approach, we have shown that this interaction is functional within the cell. PP2A/Aurora-A interaction is promoted by an S51D mutation in Aurora-A and inhibited by a phosphomimetic peptide centered around Aurora-A S51, thereby strongly suggesting that PP2A controls Aurora-A degradation by dephosphorylating serine 51 in the A box of the human enzyme.     

Direct association with inner centromere protein (INCENP) activates the novel chromosomal passenger protein, Aurora-C

A family of serine/threonine kinase Aurora constitutes a key regulator in the orchestration of mitotic events. The human Aurora paralogues Aurora-A, Aurora-B, and Aurora-C have a highly conserved catalytic domain. Extensive studies on the role of Aurora-A and Aurora-B have revealed distinct localizations and functions in regulating mitotic processes, whereas little is known about Aurora-C. The present study shows that human Aurora-C is a chromosomal passenger protein that forms complexes with Aurora-B and inner centromere protein (INCENP), which are known passenger proteins. We show that INCENP binds and activates Aurora-C in vivo and in vitro. Furthermore, Aurora-C co-expressed with INCENP elicits the phosphorylation of endogenous histone H3 in mammalian cells, even though this phosphorylation is not sufficient to establish chromosome condensation in interphase cells. We therefore suggest that Aurora-C is a novel chromosomal passenger protein that cooperates with Aurora-B to regulate mitotic chromosome dynamics in mammalian cells.     

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.     

Evolutionary relationships of Aurora kinases: Implications for model organism studies and the development of anti-cancer drugs

Background  

As key regulators of mitotic chromosome segregation, the Aurora family of serine/threonine kinases play an important role in cell division. Abnormalities in Aurora kinases have been strongly linked with cancer, which has lead to the recent development of new classes of anti-cancer drugs that specifically target the ATP-binding domain of these kinases. From an evolutionary perspective, the species distribution of the Aurora kinase family is complex. Mammals uniquely have three Aurora kinases, Aurora-A, Aurora-B, and Aurora-C, while for other metazoans, including the frog, fruitfly and nematode, only Aurora-A and Aurora-B kinases are known. The fungi have a single Aurora-like homolog. Based on the tacit assumption of orthology to human counterparts, model organism studies have been central to the functional characterization of Aurora kinases. However, the ortholog and paralog relationships of these kinases across various species have not been rigorously examined. Here, we present comprehensive evolutionary analyses of the Aurora kinase family.     

Identification of V23RalA-Ser194 as a critical mediator for Aurora-A-induced cellular motility and transformation by small pool expression screening

Human Aurora kinases have three gene family members: Aurora-A, Aurora-B, and Aurora-C. It is not yet established what the specificity of these kinases are and what signals relayed by their reactions. Therefore, we employed small pool expression screening to search for downstream substrates of Aurora-A. Interestingly, all of the identified Aurora-A substrates were resistant to serve as substrates for Aurora-B or Aurora-C, suggesting that these Aurora family members may have distinct substrate specificity for propagation of diverse signaling pathways, even though they share a conserved catalytic kinase domain. Of the candidate substrates, Aurora-A could increase the functional activity of RalA. Mutational analysis revealed that RalA-Ser194 was the phosphorylation site for Aurora-A. Ectopic expression of V23RalA-WT could enhance collagen I-induced cell migration and anchorage-independent growth in Madin-Darby canine kidney (MDCK) Aurora-A stable cell lines. In contrast, overexpression of V23RalA-S194A in MDCK Aurora-A-stable cell lines abolished the intrinsic migration and transformation abilities of Aurora-A. To our knowledge, this is the first systematic search for the downstream substrates of Aurora-A kinase. Moreover, these results support the notion that Aurora-A may act in concert with V23RalA through protein phosphorylation on Ser194 to promote collagen I-induced cell motility and anchorage-independent growth in MDCK epithelial cells.     

Searching for biomarkers of Aurora-A kinase activity: identification of in vitro substrates through a modified KESTREL approach

Aurora-A, -B, and -C are members of a small family of mitotic serine/threonine kinases that regulate centrosome maturation, chromosome segregation, and cytokinesis. They are often overexpressed in different human tumor types and have been identified as attractive targets for anticancer drug development. As specific inhibitors of the Aurora kinases are entering phase I clinical trials, there is a high need for appropriate Aurora-A biomarkers to follow mechanism of action or response. To identify novel Aurora-A substrates potentially useful as specific biomarkers we applied several modifications to the original KESTREL (Kinase Substrate Tracking and Elucidation) method in conjunction with gel electrophoresis and MALDI-MS and LC-MS/MS. The major modifications to the method included the introduction of a heating step to inactivate endogenous kinases after cell lysis and the execution of the in vitro kinase reaction in the presence of 5 mM Mg(2+) and at high (1 mM) ATP concentration. Total and fractionated extracts from nocodazole-treated HeLa cells were used as a source of Aurora-A substrates. Using this approach, we were able to detect a number of Aurora-A specific phospholabeled signals and to identify vimentin as a putative Aurora-A substrate. Vimentin was then confirmed as an in vitro substrate of Aurora-A by the phosphorylation of the recombinant protein followed by MS and antibody detection.     

Mitotic Phosphorylation of Histone H3: Spatio-Temporal Regulation by Mammalian Aurora Kinases

Phosphorylation at a highly conserved serine residue (Ser-10) in the histone H3 tail is considered to be a crucial event for the onset of mitosis. This modification appears early in the G(2) phase within pericentromeric heterochromatin and spreads in an ordered fashion coincident with mitotic chromosome condensation. Mutation of Ser-10 is essential in Tetrahymena, since it results in abnormal chromosome segregation and extensive chromosome loss during mitosis and meiosis, establishing a strong link between signaling and chromosome dynamics. Although mitotic H3 phosphorylation has been long recognized, the transduction routes and the identity of the protein kinases involved have been elusive. Here we show that the expression of Aurora-A and Aurora-B, two kinases of the Aurora/AIK family, is tightly coordinated with H3 phosphorylation during the G(2)/M transition. During the G(2) phase, the Aurora-A kinase is coexpressed while the Aurora-B kinase colocalizes with phosphorylated histone H3. At prophase and metaphase, Aurora-A is highly localized in the centrosomic region and in the spindle poles while Aurora-B is present in the centromeric region concurrent with H3 phosphorylation, to then translocate by cytokinesis to the midbody region. Both Aurora-A and Aurora-B proteins physically interact with the H3 tail and efficiently phosphorylate Ser10 both in vitro and in vivo, even if Aurora-A appears to be a better H3 kinase than Aurora-B. Since Aurora-A and Aurora-B are known to be overexpressed in a variety of human cancers, our findings provide an attractive link between cell transformation, chromatin modifications and a specific kinase system.     

Overexpression of Aurora-A enhances invasion and matrix metalloproteinase-2 expression in esophageal squamous cell carcinoma cells

Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive cancers, and metastasis is the principal cause of death in ESCC patients. It has been shown that amplification and overexpression of mitotic serine/threonine kinase Aurora-A occur in several types of human tumors, including ESCC. Moreover, increase in expression levels of Aurora-A has been predicted to correlate with the grades of tumor differentiation and invasive capability. However, the mechanisms by which Aurora-A mediates its invasive effects still remain elusive. In this article, we showed that Aurora-A overexpression significantly increased cell migration and invasion as well as secretion and expression of matrix metalloproteinase-2 (MMP-2). Conversely, siRNA-mediated knockdown of Aurora-A expression in human ESCC cells led to inhibition of cell invasiveness as well as secretion and expression of MMP-2. In addition, Aurora-A overexpression increased phosphorylation levels of p38 mitogen-activated protein kinase (MAPK) and Akt, and the knockdown of Aurora-A by siRNA decreased the activity of p38 MAPK and Akt. Moreover, the blocking of the activity of above kinases using chemical inhibitors suppressed the ability of Aurora-A to induce MMP-2 secretion and expression as well as cell invasion. These data show that overexpression of Aurora-A contributes to the malignancy development of ESCC by enhancing tumor cell invasion as well as MMP-2 activity and expression, which can occur through signaling pathways involving p38 MAPK and Akt protein kinases. Taken together, these studies provide a molecular basis for promoting the role of Aurora-A in malignancy development of ESCC.     

Overexpression of Aurora-A kinase promotes tumor cell proliferation and inhibits apoptosis in esophageal squamous cell carcinoma cell line

Attrora-A kinase, a serine/threonine protein kinase, is a potential oncogene. Amplification and overexpression of Aurora-A have been found in several types of human tumors, including esophageal squamous cell carcinoma （ESCC）. It has been demonstrated that cells overexpressing Attrora-A are more resistant to cisplatin-induced apoptosis. However, the molecular mechanisms mediating these effects remain largely unknown. In this report, we showed that overexpression of Attrora-A through stable transfection of pEGFP-Aurora-A in human ESCC KYSE150 cells significantly promoted cell proliferation and inhibited cisplatin- or UV irradiation-induced apoptosis. Cleavages of caspase-3 and poly （ADPribose） polymerase （PARP） in Attrora-A overexpressing cells were substantially reduced after cisplatin or UV treatment. Furthermore, we found that silencing of endogenous Aurora-A kinase with siRNA substantially enhanced sensitivity to cisplatin- or UV-induced apoptosis in human ESCC EC9706 cells. In parallel, overexpression of Aurora-A potently upregulated the expression of Bcl-2. Moreover, the knockdown of Bcl-2 by siRNA abrogated the Aurora-A＇s effect on inhibiting apoptosis. Taken together, these data provide evidence that Aurora-A overexpression promoting cell proliferation and inhibiting apoptosis, suggesting a novel mechanism that is closely related to malignant phenotype and anti-cancer drugs resistance of ESCC cells.     

Aurora-A and an interacting activator,the LIM protein Ajuba,are required for mitotic commitment in human cells

Aurora family kinases contribute to regulation of mitosis. Using RNA interference in synchronized HeLa cells, we now show that Aurora-A is required for mitotic entry. We found that initial activation of Aurora-A in late G2 phase of the cell cycle is essential for recruitment of the cyclin B1-Cdk1 complex to centrosomes, where it becomes activated and commits cells to mitosis. A two-hybrid screen identified the LIM protein Ajuba as an Aurora-A binding protein. Ajuba and Aurora-A interact in mitotic cells and become phosphorylated as they do so. In vitro analyses revealed that Ajuba induces the autophosphorylation and consequent activation of Aurora-A. Depletion of Ajuba prevented activation of Aurora-A at centrosomes in late G2 phase and inhibited mitotic entry. Overall, our data suggest that Ajuba is an essential activator of Aurora-A in mitotic commitment.     

Essential Roles of mTOR/Akt Pathway in Aurora-A Cell Transformation

We have recently demonstrated that Aurora-A kinase is a potential oncogene to develop mammary gland tumors in mice, when expressed under MMTV promoter. These tumors contain phosphorylated forms of Akt and mTOR, suggesting that Akt-mTOR pathway is involved in transformed phenotype induced by Aurora-A. In the present studies, we discovered that stable cell lines expressing Aurora-A contain phosphorylation of Akt Ser473 after prolonged passages of cell culture, not in cells of the early period of cell culture. Levels of PTEN tumor suppressor are significantly reduced in these late passage cells at least in part due to increased poly ubiquitination of the protein. Akt-activated Aurora-A cells formed larger colonies in soft agar and are resistant to UV-induced apoptosis. Aurora-A inhibitor, VX-680, can cause cell death of Aurora-A cells in which Akt is not activated. siRNA-mediated depletion of mTOR in those cells resulted in decreased phosphorylation of Akt Ser473, suggesting that TORC2 complex phosphorylates Akt in Aurora-A cells. Treatment of late-passage Aurora-A cells with mTOR inhibitor reduced colony formation in soft agar. These results strongly suggest that commitment of cell transformation by Aurora-A is determined by at least co-activation of Akt/mTOR pathway.     

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.     

Human TPX2 is required for targeting Aurora-A kinase to the spindle

Aurora-A is a serine-threonine kinase implicated in the assembly and maintenance of the mitotic spindle. Here we show that human Aurora-A binds to TPX2, a prominent component of the spindle apparatus. TPX2 was identified by mass spectrometry as a major protein coimmunoprecipitating specifically with Aurora-A from mitotic HeLa cell extracts. Conversely, Aurora-A could be detected in TPX2 immunoprecipitates. This indicates that subpopulations of these two proteins undergo complex formation in vivo. Binding studies demonstrated that the NH2 terminus of TPX2 can directly interact with the COOH-terminal catalytic domain of Aurora-A. Although kinase activity was not required for this interaction, TPX2 was readily phosphorylated by Aurora-A. Upon siRNA-mediated elimination of TPX2 from cells, the association of Aurora-A with the spindle microtubules was abolished, although its association with spindle poles was unaffected. Conversely, depletion of Aurora-A by siRNA had no detectable influence on the localization of TPX2. We propose that human TPX2 is required for targeting Aurora-A kinase to the spindle apparatus. In turn, Aurora-A might regulate the function of TPX2 during spindle assembly.     

Two newly identified sites in the N-terminal regulatory domain of Aurora-A are essential for auto-inhibition

Aurora-A, a centrosome-localized serine/threonine kinase, is over-expressed in multiple human cancers. We previously reported Zhang et al. (Biochem Biophys Res Commun 2007, 357:347–352) intramolecular inhibitory regulation of Aurora-A between its N-terminal (Nt) regulatory domain (amino acids 1–128, Nt) and C-terminal catalytic domain (aa 129–403, Cd). Here, we identified two essential sites located on the Nt of Aurora-A (Lys 99 and Lys 119) and demonstrate that mutation of either residue to Gly could cause the Nt and C-terminal lobes of the catalytic domain in Aurora-A to form a closed conformation, resulting in a loss of kinase activity. This inactive conformation was reversed by adding C26 peptide (274–299) or Ajuba, which is a required activator of Aurora-A. Over-expression of either mutant induced G2/M arrest. These results provide a basis for future anti-cancer studies targeting Aurora-A.     

Phosphorylation and Activation of Androgen Receptor by Aurora-A

Aurora-A kinase is frequently overexpressed/activated in various types of human malignancy, including prostate cancer. In this study, we demonstrate elevated levels of Aurora-A in androgen-refractory LNCaP-RF but not androgen-sensitive LNCaP cells, which prompted us to examine whether Aurora-A regulates the androgen receptor (AR) and whether elevated Aurora-A is involved in androgen-independent cell growth. We show that ectopic expression of Aurora-A induces AR transactivation activity in the presence and absence of androgen. Aurora-A interacts with AR and phosphorylates AR at Thr²⁸² and Ser²⁹³ in vitro and in vivo. Aurora-A induces AR transactivation activity in a phosphorylation-dependent manner. Ectopic expression of Aurora-A in LNCaP cells induces prostate-specific antigen expression and cell survival, whereas knockdown of Aurora-A sensitizes LNCaP-RF cells to apoptosis and cell growth arrest. These data indicate that AR is a substrate of Aurora-A and that elevated Aurora-A could contribute to androgen-independent cell growth by phosphorylation and activation of AR.     

Structures of the cancer-related Aurora-A,FAK, and EphA2 protein kinases from nanovolume crystallography

Protein kinases are important drug targets in human cancers, inflammation, and metabolic diseases. This report presents the structures of kinase domains for three cancer-associated protein kinases: ephrin receptor A2 (EphA2), focal adhesion kinase (FAK), and Aurora-A. The expression profiles of EphA2, FAK, and Aurora-A in carcinomas suggest that inhibitors of these kinases may have inherent potential as therapeutic agents. The structures were determined from crystals grown in nanovolume droplets, which produced high-resolution diffraction data at 1.7, 1.9, and 2.3 A for FAK, Aurora-A, and EphA2, respectively. The FAK and Aurora-A structures are the first determined within two unique subfamilies of human kinases, and all three structures provide new insights into kinase regulation and the design of selective inhibitors.