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Genes to Cells (2005) 10, 627-638. doi:10.1111/j.1365-2443.2005.00870.x
© 2005 Blackwell Publishing or its licensors
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Over-expression of Aurora-A targets cytoplasmic polyadenylation element binding protein and promotes mRNA polyadenylation of Cdk1 and cyclin B1

Takashi Sasayama1,2, Tomotoshi Marumoto1,2, Naoko Kunitoku1, Dongwei Zhang1, Norihiko Tamaki2, Eiji Kohmura2, Hideyuki Saya1 and Toru Hirota1,a,*

1 Department of Tumor Genetics and Biology, Graduate School of Medical Sciences, Kumamoto University, 1-1-1 Honjo, Kumamoto 860-8556, Japan
2 Department of Neurosurgery, Kobe University Graduate School of Medicine, 7-5-1, Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 Supplementary Material
 References
 
Aurora-A is a centrosomal serine-threonine kinase that regulates mitosis. Over-expression of Aurora-A has been found in a wide range of tumors and has been implicated in oncogenic transformation. However, how Aurora-A over-expression contributes to promotion of carcinogenesis remains elusive. Immunohistochemical analysis of breast tumors revealed that over-expressed Aurora-A is not restricted to the centrosomes but is also found in the cytoplasm. This over-expressed Aurora-A appeared to be phosphorylated on Thr288, which is known to be required for its enzymatic activation. In analogy to Aurora-A's role in oocyte maturation and the early embryonic cell cycle, here we investigated whether ectopically over-expressed Aurora-A can similarly stimulate polyadenylation of mRNA in human somatic cultured cells by interacting with a human ortholog of cytoplasmic polyadenylation element binding protein, h-CPEB. In vitro experiments revealed that Aurora-A binds directly to, and phosphorylates, h-CPEB. We found that polyadenylation of mRNA tails of cyclin B1 and Cdk1 was synergistically stimulated when Aurora-A and h-CPEB were over-expressed, and they were further promoted in the presence of an Aurora-A activator Ajuba. Our results suggest a function of ectopically over-expressed Aurora-A that might be relevant for carcinogenesis.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 Supplementary Material
 References
 
Aurora-A is a mitotic kinase that controls centrosome maturation, mitotic entry, spindle formation and chromosome alignment. Apart from these multiple physiological roles of Aurora-A in mitosis, several lines of evidence indicate that Aurora-A is an oncogene that has medical relevance (reviewed in Dutertre et al. 2002; Meraldi et al. 2004; Marumoto et al. 2005).

Over-expression of Aurora-A has been reported in a range of primary tumors, including that of breast (Tanaka et al. 1999), colon and rectum (Takahashi et al. 2000), ovary (Gritsko et al. 2003), pancreas (Li et al. 2003), as well as in many cancer-derived cell lines (reviewed in Dutertre et al. 2002). The human Aurora-A gene localizes to chromosome 20q13.2, a region frequently amplified in various malignant tumors. Accordingly, gene amplification of Aurora-A is associated with its elevated protein levels in many cancers (Sen et al. 1997; Bischoff et al. 1998; Zhou et al. 1998; Tanner et al. 2000). It is noteworthy that increased levels of Aurora-A mRNA correlate with chromosome instability in tumors (Miyoshi et al. 2001). Observations in cultured cells indicated that Aurora-A protein levels increase periodically during the cell cycle, at G2 to M phase, and the protein is enriched at centrosomes and mitotic spindles (Kimura et al. 1997; Bischoff et al. 1998; Marumoto et al. 2002). In contrast to these cytological characterizations, immunohistochemical analyses of the tumors reveal that over-expressed Aurora-A can often be found rather homogenously within the lesions and its intracellular localization is not confined to the centrosomes but also accumulates in the cytoplasm (Tanaka et al. 1999; Takahashi et al. 2000; Gritsko et al. 2003).

The oncogenic activity of Aurora-A has been well documented. Rodent fibroblasts transfected with wild-type Aurora-A cDNA can grow and form colonies in soft agar media. When these cells were inoculated into athymic mice, they develop into tumors (Bischoff et al. 1998; Zhou et al. 1998). Cytological analyses revealed that over-expression of Aurora-A results in centrosome amplification, cytokinesis failure, and thus production of aneuploid cells (Zhou et al. 1998; Meraldi et al. 2002). Moreover, exogenous expression of Aurora-A promoted cells to abrogate G2 arrest induced by DNA damage (Marumoto et al. 2002). All of these experimental and clinical observations have led to an assumption that over-expression of Aurora-A might be primarily involved in propagation of chromosome instability and in carcinogenesis.

Aurora-A is also known to be involved in meiotic cell cycle progression during oocyte maturation, which requires translation of several maternal mRNAs (reviewed in Mendez & Richter 2001). A specific RNA sequence in the 3' untranslated region of mRNA called the cytoplasmic polyadenylation element (CPE) and its acting protein, CPE binding protein (CPEB), play a crucial role in such process (Stebbins-Boaz et al. 1996). In progesterone-stimulated Xenopus oocytes, the best characterized substrate for Aurora-A's kinase activity is CPEB (Hake & Richter 1994; Mendez et al. 2000a; Tay et al. 2003). Phosphorylation of CPEB by Aurora-A facilitates recruitment of CPSF (cleavage and polyadenylation specific factor) and PAP (poly(A) polymerase) and stimulates polyadenylation/translation of meiotic cell cycle regulators (Mendez et al. 2000a,b). Furthermore, it has been shown that Aurora-A-CPEB mediates polyadenylation in the early embryonic cell cycle likewise in meiotic progression (Groisman et al. 2002). Importantly, extracts from mitotic mammalian cells retain a cytoplasmic polyadenylation activity, leading to the idea that CPE-mediated translational control might be a shared feature between meiosis and mitosis (Groisman et al. 2002).

Analogous to the physiological link between Aurora-A and CPEB, we tested whether ectopically expressed Aurora-A can target CPEB, and if this interaction can stimulate polyadenylation of mRNAs in mammalian somatic cultured cells. We examined the poly(A) length of several candidate mRNAs and found that poly(A) tails of cyclin B1 and cyclin dependent kinase 1 (Cdk1) mRNA were synergistically elongated by Aurora-A and CPEB. Our observations suggest that an aberrant over-expression of Aurora-A may stimulate mRNA polyadenylation of mitotic regulators in tumors.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 Supplementary Material
 References
 
Localization of Aurora-A and CPEB in cancer cells

Immunohistochemical analysis of breast tumors revealed that Aurora-A is over-expressed in all four samples examined, and intense signals for Aurora-A were found predominantly in the cytoplasm (Fig. 1A), in agreement with previous observations (Tanaka et al. 1999; Takahashi et al. 2000; Gritsko et al. 2003). Similarly, in cultured cells, the ectopically expressed Aurora-A distributed throughout the cytoplasm in most of the transfected cells (Fig. 1B). Using specific antibodies to Aurora-A phosphorylated on Thr-288 (T288P) in microscopy, this Aurora-A accumulated in cytoplasm appears to be phosphorylated on Thr288 (Fig. 1A). As this modification is required for its enzymatic activity (Walter et al. 2000; Littlepage et al. 2002), it was tempting to postulate that a cytoplasmic protein may serve as a substrate for over-expressed Aurora-A. In light of Aurora-A's function in oocyte maturation and in early embryonic cell cycle (reviewed in Mendez & Richter 2001), CPEB was a candidate for such a substrate.



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  		Figure 1  Expression and localization of Aurora-A and CPEB in cancer cells. (A) Immunohistochemical studies of Aurora-A in breast tumor. The frozen sections were prepared from invasive ductal breast carcinomas and immunostained with anti-Aurora-A polyclonal antibodies (left column), or with anti-phospho-Aurora-A (T288P) polyclonal antibodies (right column). Note that high levels of Aurora-A with phosphorylation on Thr288 were found in the cytoplasm. (B) Cytoplasmic distribution of exogenously expressed Aurora-A. HEK293T cells were transfected with plasmid encoding Flag-tagged Aurora-A cDNA, and 48 h after the transfection, cells were fixed and stained with anti-Flag antibodies and labeled with FITC (upper panel). 100 transfected cells were scored for the distribution of Flag-Aurora-A protein; nuclear dominant (N > C), cytoplasmic dominant (N < C) or equally distributed (N = C) (lower panel). (C) Northern blot analysis of h-CPEB in human cancer cell lines. A membrane transferred with 40 µg of electrophoresed total RNA from a human cancer cell was hybridized with 32P-labeled h-CPEB DNA probe. The single 3.3 kb transcript is marked. Ethidium bromide staining for detection of 18S rRNA of the total RNA is shown as a loading control. (D,E) Subcellular localization of h-CPEB. (D) HeLa cells were transfected with plasmid encoding GFP-h-CPEB, and GFP fluorescence (green) with DNA which was stained with propidium iodide (red) and their merged picture is shown (upper panels). NIH3T3 cells were co-transfected with HA-h-CPEB and Flag-Aurora-A, and immunostained with anti-HA (green) or anti-Flag monoclonal antibodies (red) and merged picture is shown (lower panels). Scale bar indicates 10 µm. (E) MM-LM, HeLa and MCF cells were fixed and stained with polyclonal antibodies to h-CPEB and labeled with FITC (green). DNA was counterstained with propidium iodide, PI (red). Scale bar indicates 10 µm.

 
A human ortholog of CPEB, h-CPEB, has been identified, and its expression was not abundant in many organs (Welk et al. 2001). We examined h-CPEB mRNA levels in several tumor cell lines, and found that 50% of these lines showed high expression of h-CPEB, but little or no expression of h-CPEB mRNA was observed in several cell lines including MCF-7, HCT116, and SW480 (Fig. 1C). Cell lines with high h-CPEB expression were not necessarily derived from organs in which expression of h-CPEB was detected (data not shown). To examine the localization of h-CPEB, cells were transfected with a plasmid encoding h-CPEB tagged with either enhanced green fluorescent protein (EGFP) or the hemagglutinin (HA) epitope. Most of the transfected cells revealed cytoplasmic localization of the tagged h-CPEB protein, and this distribution was not detectably affected upon co-expression with Aurora-A (Fig. 1D). Consistently, h-CPEB antibodies predominantly stained cytoplasm of MM-LM and HeLa cells (Fig. 1E). With these antibodies h-CPEB was hardly detectable in MCF-7 cells, whose expression was undetectable by Northern blot analysis (Fig. 1C). In addition, breast tumor specimens also showed cytoplasmic staining with the h-CPEB antibodies in the cancerous lesion (data not shown). These results suggest that a fraction of cancer cells have elevated levels of h-CPEB protein, which may mostly be deposited in the cytoplasm.

Interaction of h-CPEB with Aurora-A

To examine if Aurora-A interacts with h-CPEB, human embryonic kidney (HEK) 293T cells were transiently expressed with Flag-Aurora-A and HA-h-CPEB. When cell lysate co-expressing Flag-Aurora-A and HA-h-CPEB was immunoprecipitated with the anti-Flag antibodies, HA-h-CPEB was detected in the Flag-Aurora-A immune complex. Conversely, Flag-Aurora-A was detected in the HA-h-CPEB immune complex (Fig. 2A).



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  		Figure 2  Interaction of h-CPEB with Aurora-A. (A) Interaction of ectopically expressed h-CPEB with Aurora-A in HEK293T cells. Total extracts of cells expressing Flag-Aurora-A, HA-h-CPEB or both proteins were subjected to immunoblot analysis with antibodies to Flag or to HA, as indicated (bottom panels). The same cell lysates were subjected to immunoprecipitation with antibodies to HA or to Flag epitopes and the resulting precipitates were subjected to immunoblot analysis with antibodies to Flag or HA (upper and middle panels). Note that both Flag-Aurora-A (Fig. 1B) and HA-h-CPEB (data not shown) distributed diffusely in cytoplasm. (B) Interaction of maltose binding protein (MBP)-fused h-CPEB and His6-Aurora-A in an in vitro binding assay. MBP-h-CPEB protein or MBP were incubated either with His6-wild-type (WT) or kinase-inactive mutant of Aurora-A (KD), or without Aurora-A (–), and then precipitated with amylose resin beads. Bead-bound proteins were subjected to immunoblot analysis with antibodies to Aurora-A. (C) Schematic representation of wild-type and deletion mutants of Aurora-A. (D) Interaction between h-CPEB and deletion mutants of Aurora-A. Lysates of cells expressing wild-type or deletion mutants ({Delta}C1 and {Delta}C2) of Flag-Aurora-A and HA-h-CPEB were subjected to immunoprecipitation as in (A).

 
Moreover, we performed an in vitro binding assay using purified recombinant proteins, maltose binding protein (MBP)-fused h-CPEB and recombinant hexahistidine (His6) tagged Aurora-A. This binding assay suggested that Aurora-A directly binds to h-CPEB (Fig. 2B). Interestingly, the amount of co-precipitated His6-Aurora-A was more abundant when the kinase-inactive mutant of Aurora-A was used, raising a possibility that a stable association between Aurora-A and h-CPEB is affected by enzymatic activity of Aurora-A. Consistent with this idea, MBP-h-CPEB was found to associate with the fastest mobility form of His6-Aurora-A, which presumably is an inactive form of Aurora-A (Fig. 2B, lanes 10 and 11).

To identify the binding site for h-CPEB in Aurora-A, we generated deletion mutants of Flag-Aurora-A (Fig. 2C), and immunoprecipitation experiments were performed with these mutants. Wild-type Aurora-A and {Delta}C1 mutant of Aurora-A which lacks 20 amino acids of its carboxyl terminus were still able to associate with HA-h-CPEB; however, the {Delta}C2 mutant which does not contain the catalytic domain failed to bind to HA-h-CPEB (Fig. 2D), suggesting that the catalytic domain of Aurora-A is necessary for their interaction.

h-CPEB serves as a substrate for Aurora-A

The observation that the interaction of Aurora-A with h-CPEB was more stable with kinase inactive Aurora-A (Fig. 2B) led us to ask if h-CPEB serves as a substrate for Aurora-A, as is characterized in Xenopus and mouse oocytes (Mendez et al. 2000a; Hodgman et al. 2001). An in vitro kinase assay was performed using recombinant GST-fused wild-type Aurora-A or a kinase-inactive mutant thereof (K162M) purified from baculovirus-infected Sf9 cells and recombinant His6-tagged h-CPEB protein generated in bacteria. Phosphorylation of h-CPEB was observed on incubation with the wild-type Aurora-A, but not with the kinase inactive mutant (Fig. 3A).



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  		Figure 3  Phosphorylation of h-CPEB by Aurora-A. (A) Phosphorylation of h-CPEB by Aurora-A in vitro. Purified His6-h-CPEB was incubated for 30 min with purified GST-Aurora-A, either wild-type (WT) or the kinase-inactive mutant (KD), in the presence of [{gamma}-32P]ATP. Incorporation of 32P was then visualized by SDS-PAGE and autoradiography. (B) Change in electrophoretic mobility of CPEB by co-expressing Aurora-A. MCF-7 cells were transfected with a combination of an expression plasmid for Flag-h-CPEB, HA-Aurora-A (wild-type or kinase-inactive mutant) and Myc-Ajuba, as shown in the panel. Cell extracts were subjected to immunoblot analysis with antibodies to Flag, HA, or Myc, as indicated. (C) Phosphorylation of Flag-h-CPEB. Extracts of MCF7 cells expressing Flag-h-CPEB, HA-Aurora-A and Myc-Ajuba were incubated with or without 5 unit of calf intestinal alkaline phosphatase (CIAP) and 50 mMß-glycerophosphate for 2 h at 37 °C, as indicated. The extracts were then subjected to immunoblot analysis with anti-Flag monoclonal antibody. (D) Phosphorylation of h-CPEB in the in-gel kinase assay. Total cell extracts prepared from SW480, HCT15, Colo205, U251, MCF-7 and MM-LM cell lines were tested for h-CPEB phosphorylating activity in gels containing 25 µg/mL of GST-h-CPEB (first panel) or 100 µg/mL of GST (second panel). In parallel, extracts were subjected to immunoblot analysis (IB) with antibodies to Aurora-A (third panel), or {alpha}-tubulin (fourth panel) as a loading control.

 
Previous experiments showed that kinase activity remain at low levels when wild-type Aurora-A alone was transfected, but that kinase activity was increased when Ajuba, an activator for Aurora-A, was co-expressed (Hirota et al. 2003). A fraction of h-CPEB resulted in a slower migrating form when HA-Aurora-A was co-expressed with Myc-tagged Ajuba, whereas the expression of the wild-type Aurora-A alone or the kinase inactive Aurora-A did not induce the slower migrating form (Fig. 3B). The slowest migrating form of Flag-h-CPEB was converted to the faster form by phosphatase treatment in a manner that was sensitive to ß-glycerophosphate (Fig. 3C), indicating that the slowly migrating form corresponds to phosphorylated h-CPEB. These results suggest that h-CPEB can serve as a substrate of Aurora-A in cells.

To further verify Aurora-A as a kinase that phosphorylates h-CPEB in tumor cells, an in-gel kinase assay was performed. Equal amounts of total cell extracts from several cancer cells were resolved by SDS-PAGE using recombinant GST-h-CPEB containing gel, renatured and incubated in the presence of [{gamma}-32P]ATP. The presence of active kinase for h-CPEB can be indicated as radioactive bands representing phosphorylated GST-h-CPEB. We found a major radioactive band migrating at ~48 kDa, which was compatible with the molecular weight of Aurora-A (Fig. 3D upper panel). The appearance of the 48 kDa band was not likely to result from autophosphorylation of the kinase nor phosphorylation of GST, because this band did not appear with a GST-mock containing gel (Fig. 3D second panel). Marked h-CPEB phosphorylation activity was observed in SW480 cell extract, which has a high level of Aurora-A expression; but no activity was found with Colo205 cells in which Aurora-A was undetectable (Fig. 3D third panel). These correlations support the assertion that the radioactive band at ~48 kDa is dependent on Aurora-A.

To determine the phosphorylation sites in h-CPEB, we first generated both carboxyl terminal-truncated ({Delta}C) and amino terminal-truncated ({Delta}N) mutants of h-CPEB (Fig. 4A). The phosphorylation of h-CPEB was clearly stronger in the {Delta}C mutant than the other (Fig. 4B). The amino acid sequence of h-CPEB ({Delta}C) revealed two leucine-rich regions and two serine/threonine-rich regions, and thus we divided h-CPEB ({Delta}C) further into five polypeptides (Fig. 4A). The major phosphorylation site(s) appeared to reside in the {Delta}C-4 fragment that consisted of amino acids 91–122 (Fig. 4C). This region includes two Aurora-A phosphorylation motifs Leu-Asp-Ser/Thr-Arg (LDS/TR), which have been characterized in Xenopus and mouse CPEB (Mendez et al. 2000a; Tay et al. 2003). However, when we changed Thr/Ser to Ala within these LDS/TR motifs, phosphorylation of the {Delta}C-4 fragment resulted in only a slight reduction, suggesting that Aurora-A can also phosphorylate other site(s) than the LDS/TR motif in vitro (data not shown).



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  		Figure 4  Phosphorylation sites of h-CPEB by Aurora-A. (A) Schematic representation of wild-type and mutants of h-CPEB. Domain organization in h-CPEB protein is shown as follows: CHD2, 3: CPEB homology domain 2, 3; RRM1, 2: RNA recognition motif 1, 2; Zn-f: Zinc-finger domain; PEST: PEST sequence. The N-terminal half of hCPEB, h-CPEB{Delta}C, were further divided into five polypeptides; {Delta}C1 (aa1–30), {Delta}C2 (aa31–51), {Delta}C3 (aa52–90), {Delta}C4 (aa91–122) and {Delta}C5 (aa123–157). {Delta}C1 and {Delta}C5 contain leucine rich (L rich) sequence, and {Delta}C2 and {Delta}C4 contain many serine or threonine residues (S/T rich). Amino acid sequence of {Delta}C4 fragment is shown. Two Aurora phosphorylation motifs are underlined, and PEST sequence is boxed. Asterisks mark serine and threonine. (B) Phosphorylation of deletion mutants of h-CPEB by Aurora-A in vitro. Purified GST, GST-h-CPEB {Delta}N or {Delta}C were incubated with purified His6-Aurora-A of either wild-type (WT) or a kinase inactive mutant (KD) in the presence of [{gamma}-32P]ATP, and incorporation of 32P was then analyzed by SDS-PAGE and autoradiography (upper panel). The gel was also stained with Coomassie Brilliant Blue (CBB) (lower panel). (C) Phosphorylation of h-CPEB {Delta}C mutants by Aurora-A. GST-CPEB {Delta}C mutants ({Delta}C1~5) were incubated with His6-Aurora-A in the presence of [{gamma}-32P]ATP and analyzed as in (B) (upper panel). The gel was also stained with Coomassie Brilliant Blue (CBB) (lower panel).

 
Aurora-A and h-CPEB stimulate polyadenylation of cyclin B1 and Cdk1 mRNA

In oocytes and in early embryonic cells, Aurora-A stimulates polyadenylation of several mRNAs by phosphorylating CPEB (Mendez et al. 2000a; Groisman et al. 2002). To examine if over-expression of Aurora-A can induce mRNA polyadenylation similarly in mammalian somatic cells, Rat1 fibroblasts with high expression levels of Aurora-A were analyzed for the poly(A) length of several mRNAs. To measure poly(A) length, we followed the method called the poly(A) tail length (PAT) assay, which was originally developed by Salles et al. (1992). Among CPEB targets known in the oocyte (Stebbins-Boaz et al. 1996; de Moor & Richter 1999; Mendez et al. 2000a), we first focused on the poly(A) length of cyclin A2, cyclin B1, Cdk1 and Cdk2 mRNAs. While the poly(A) tail length of cyclin A2 and Cdk2 were not detectably affected by Aurora-A expression, that of cyclin B1 and Cdk1 were extended upon expression of Aurora-A (Fig. 5A). The extension of poly(A) seemed to require kinase activity of Aurora-A, since it was not extended by expressing kinase-inactive Aurora-A (Fig. 5A). We compared the cell cycle distributions of these cell lines by flow cytometric analysis, which suggested that elongation of poly(A) tails was not due to mitotic enrichment (Supplementary Fig. S1).



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  		Figure 5  Analysis of poly(A) length. (A) Poly(A) tail extension in cells with exogenous expression of Aurora-A. Total RNA were extracted from Rat1 cells (Parental cell), or Rat1 cells that stably express Aurora-A wild-type (WT) or the kinase inactive form (KD). A PAT assay was performed using specific primers to rat Cdk1, Cdk2, cyclin B1 and cyclin A2. The products were resolved on 3% agarose gel and visualized by ethidium bromide staining. The sizes of their poly(A) tails are indicated. The amounts of 28S rRNA verify the total RNA used in the PAT assay (bottom panel). Similar results were obtained in three independent experiments. (B) Enhancement of poly(A) extension by expression of h-CPEB and Aurora-A. MCF-7 cells were transfected with the indicated combination of plasmid DNA encoding Flag-h-CPEB, HA-Aurora-A(WT), HA-Aurora-A(KD), and Myc-Ajuba. Following RNA extraction, a PAT assay was performed using specific primers to indicated proteins. Note that poly(A) length of Cdk1 and cyclin B1 mRNA were synergistically elongated by Aurora-A(WT) and CPEB expression, while poly(A) lengths of Cdk2, cyclin A2, cyclin E1, cyclin D3 or ß-actin were not detectably affected. Similar results were obtained in three independent experiments.

 
To examine the involvement of h-CPEB in the Aurora-A-induced poly(A) extension, we utilized MCF-7 cells, whose endogenous CPEB expression was not detectable in Northern blot analysis (Fig. 1C). Upon expression of h-CPEB, the poly(A) lengths of both Cdk1 and cyclin B1 mRNA were extended (Fig. 5B, lane 2). When Aurora-A was expressed in combination with h-CPEB, the polyadenylation was synergistically stimulated (Fig. 5B, lane 3). Poly(A) extension was further promoted in the presence of an Aurora-A activator, Ajuba (Fig. 5B, lane 4). Similar results were obtained in Rat1 fibroblasts (Supplementary Fig. S2A). For Cdk1 polyadenylation in MCF cells, it might be possible to infer that the kinase inactive Aurora-A shows dominant negative effect, since the extension of poly(A) of Cdk1 seems to be suppressed by Aurora-A (KD) expression (compare lanes 5, 6 and 1 in Fig. 5B).

These data suggest that ectopic expression of Aurora-A and h-CPEB can stimulate polyadenylation of cyclin B1 and Cdk1, which is the primary kinase driving mitosis. We have analyzed a wide range of mRNAs that contain the CPEB binding motif at their 3' untranslated region and whose encoded protein is involved in cell proliferation, apoptosis or translational activation (Supplementary Fig. S2B). However, cyclin B1 and Cdk1 were the only mRNAs in which polyadenylation was found to be stimulated by Aurora-A and h-CPEB.


   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 Supplementary Material
 References
 
In light of Aurora-A's role in oocyte maturation and in the early embryonic cell cycle, we examined if ectopically over-expressed Aurora-A can similarly target h-CPEB and stimulate polyadenylation of mRNA in human carcinoma cells. Among tested candidates, we found that Aurora-A and h-CPEB synergistically elongate the poly(A) tails of cyclin B1 and Cdk1 mRNA. Our results suggest a molecular pathway which might be stimulated by elevated levels of Aurora-A in cancer cells.

Immunohistochemical analysis of breast tumors suggests that regulation of Aurora-A seems to be impaired for both protein localization and enzymatic activation (Tanaka et al. 1999; Takahashi et al. 2000; Gritsko et al. 2003; Fig. 1A). In normal mitosis, the enzymatic activity of Aurora-A is known to be controlled by several binding proteins, including TPX2 (Kufer et al. 2002) and Ajuba (Hirota et al. 2003). It is therefore possible that these Aurora-A binding proteins could stimulate the kinase activity of Aurora-A when they are also overproduced. Notably, the mRNA expression of both TPX2 and Ajuba is found to be up-regulated in a number of malignancies (Manda et al. 1999; T. S and T. H., unpublished observations). Alternatively, activation of Aurora-A could also be due to a shortage of inhibitory proteins. A physiological protein concentration of negative regulators for Aurora-A, such as type1 protein phosphatase (Katayama et al. 2001), might not be enough to inhibit all of the overproduced Aurora-A molecules, resulting in the deposition of active enzyme. Intriguingly, p53 protein has been shown to suppress the kinase activity of Aurora-A (Eyers & Maller 2004), which might explain why Aurora-A becomes active in cancers without functional p53.

Our results imply that the human ortholog of CPEB, h-CPEB, can be a relevant substrate for over-expressed Aurora-A which accumulates in the cytoplasm. Having known that h-CPEB is not ubiquitously expressed among organs, this pathway could be particularly significant in cancers that associate with elevated levels of h-CPEB expression (Fig. 1C). Moreover, it is interesting to know whether the interaction between Aurora-A and CPEB is similarly involved in mitotic control for somatic cells as in meiosis or in early developmental stages. However, the observation that CPEB knockout mice are viable but revealed male infertility may imply that the involvement of CPEB in mitotic control is not absolutely required in a physiological context within somatic cells(Tay & Richter 2001).

Among several candidates, we found that polyadenylation of cyclin B1 and Cdk1 is stimulated by Aurora-A and h-CPEB expression (Fig. 5). The expression levels of cyclin B1 are strictly regulated by both transcription and proteolysis during the cell cycle, and are therefore confined to G2-M phase. However, several immunohistochemical studies have shown that the cyclin B1 levels are elevated in a significant fraction of malignant tumors (Soria et al. 2000; Yasuda et al. 2002). Various causes might account for this, but abnormally long-lived mRNA can be attributable as reported by Keyomarsi & Pardee (1993). By analyzing cultured cells derived from breast tumors, they found that over-expression of cyclin B1 and Cdk1 in cancer cells is accompanied by an increased stability of the mRNA. It is intriguing to examine if the poly(A) are extended and if activation of Aurora-A-CPEB pathway could contribute to the increased stability of the mRNA in those cell lines.

What could be the consequences when expression of cyclin B1 and Cdk1 are stimulated by Aurora-A?

Because cyclin B1 and Cdk1 forms a complex for driving the G2-M transition and an elevated level of cyclin B1 by itself is known to override the G2 arrest (Innocente et al. 1999), over-expression of Aurora-A might strongly promote mitotic entry, similarly to the egg maturation process in frogs (Andresson & Ruderman 1998). This idea is consistent with the observation that G2 arrest induced by DNA damage was abrogated by Aurora-A over-expression (Marumoto et al. 2002). Moreover, in mitosis, Aurora-A over-expression is found to interfere with the spindle assembly checkpoint (Anand et al. 2003). Since a functional screen in budding yeast revealed that over-expression of cyclin B1 facilitates spindle assembly checkpoint abrogation (Sarafan-Vasseu et al. 2002), the increased cyclin B1 translation induced by Aurora-A and h-CPEB might be sufficient for this phenomenon.

A number of models have been proposed to explain how over-expressed Aurora-A leads cells to malignant transformation. Given that Aurora-A is a centrosomal protein and regulates centrosome function and mitotic spindle organization, it is conceivable that elevated levels of Aurora-A can induce mitotic failures (Meraldi et al. 2004). Our results suggest the possibility that the Aurora-A–h-CPEB pathway can lead to over-expression of cyclin B1 and Cdk1, which might contribute to overriding G2 and/or mitotic checkpoints. All of these aberrations in the control of G2-M phase may give rise to chromosome instability and contribute to rapid accumulation of genetic abnormalities. A screen of gene expression that is promoted by Aurora-A-h-CPEB pathway could provide further clues to elucidate the underlying mechanism by which Aurora-A triggers oncogenic transformation.


   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
Supplementary Material
 References
 
Cell culture and transfection

HeLa, HEK293T, MCF-7, U251, MM-LM and Rat-1 cells were cultured in DMEM/F12 medium supplemented with 10% fetal calf serum without antibiotics. For transfection into HeLa, MCF-7 or Rat-1, cultured cells were seeded on to 6-well plates and transfected using FuGene6 transfection reagent following the manufacturer's instruction (Roche). For HEK293T or NIH3T3 cells, Lipofectamine and PLUS reagent (Invitrogen) were used for transfections.

Plasmids

The open reading frame of h-CPEB, Aurora-A and Ajuba were amplified by PCR from the HeLa cDNA library. All PCR products were obtained using Pyrobest DNA polymerase (TaKaRa) and confirmed by DNA sequencing. Two transcripts of h-CPEB, referred to as the long form and the short form, whose encoded protein lacks initial 75 amino acids, have been identified (Welk et al. 2001). However, because the short form is namely expressed in various cancer cell lines (data not shown) and includes a consensus site for Eg2/Aurora-A phosphorylation (Mendez et al. 2000a), we studied the interaction between the short form of h-CPEB and Aurora-A. To generate glutathione-S-transferase (GST), hexahistidine (His6)-tagged, or maltose binding protein (MBP)-tagged proteins, PCR fragments were subcloned into pGEX4T-1 (Amersham Pharmacia Biothech), pRSET (Invitrogen) or pMAL-c2X (New England BioLab), respectively. For protein expression in Sf9 insect cells, DNA fragments were cloned into pFastBac (Gibco BRL).

Antibodies

Rabbit polyclonal antibodies against Aurora-A were generated as previously described (Marumoto et al. 2002). Antibodies specific for the phospho-Thr288 form of Aurora-A were purchased from Cell Signaling Technology. Polyclonal antibodies to CPEB were purchased from Affinity BioReagents. Monoclonal antibodies for Flag-epitope (M5), HA-epitope (3F10), and Myc-epitope (PL14) were obtained from Sigma, Roche, and Medical & Biological Laboratories, respectively. Mouse monoclonal antibodies to {alpha}-tubulin (B512) were obtained from Sigma.

Purification of recombinant proteins

Recombinant baculoviruses, His6-tagged and GST-fused recombinant proteins were produced as described previously (Hirota et al. 2003; Kunitoku et al. 2003). For purification of MBP-h-CPEB protein, Escherichia coli (BL21-SI) containing expression plasmids for MBP-h-CPEB were grown in LB broth at 20 °C for 24 h, transferred to 15 °C, and were induced with 1 mM isopropyl-1-thio-ß-D-galactopyranoside (IPTG) for 12 h. Cells were resuspended in cold column buffer (20 mM Tris-Cl (pH 8.0), 200 mM NaCl, 1 mM EDTA, 10 mMß-mercaptoethanol, 1 mM DNase, 1.0% Triton X-100, and protease inhibitors). It was then sonicated six times (15 s each) on ice and centrifuged. Soluble proteins were then subjected to chromatography with amylose resin columns (New England Biolabs); elution was performed with the column buffer containing 10 mM maltose (pH 8.0).

In vitro and in-gel kinase assay

The in vitro kinase assay was performed as described (Hirota et al. 2003). For in-gel kinase assay, cells were lyzed in lysis buffer (20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% NP-40, 10% Glycerol, 0.5 mM EDTA, 0.5 mM EGTA, 20 mMß-glycerophosphate, and protease inhibitors). Fifty µg of cell extracts were electrophoresed in 12% SDS-polyacrylamide gels containing 0.025 mg/mL of GST-h-CPEB or 0.1 mg/mL of GST protein. The gels were washed in 50 mM HEPES-NaOH (pH 7.5) containing 20% isopropanol for 1 h and denatured by two washes in buffer A (50 mM HEPES [pH 7.5], 5 mMß-mercaptoethanol) and two washes in buffer A containing 6 M guanidine hydrochloride. Step-wise renaturation was performed with buffer A containing 0.05% Tween 20, with a final overnight wash in the same buffer. Kinase reactions were performed in kinase buffer (20 mM HEPES-NaOH (pH 7.5), 20 mM MgCl2, 2 mM DTT, 50 µM ATP, 50 µCi of [{gamma}-32P]ATP/mL) for 30 min at 30 °C, and the gels were washed several times with a mixture containing 5% trichloroacetic acid and 1% sodium pyrophosphate, dried, and detected by autoradiography.

Immunoprecipitation

For immunoprecipitation, cells transfected with the vectors were lyzed on ice for 30 min with lysis buffer containing 1.0% NP-40, 20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 5% glycerol, 1 mM DTT, 20 mMß-glycerophosphate, 1 mM sodium vanadate, 2 µg/mL aprotinin, 1 mM 4-(2-Aminoethyl) benzenesulfonyl fluoride (AEBSF), 10 µM leupeptin, and 1 mM pepstatin. Lysates were centrifuged at 14 000 g for 30 min. Aliquots of supernatant were incubated for 2 h at 4 °C with anti-Flag or anti-HA antibodies conjugated to agarose beads. After washing the beads with a washing buffer containing 0.5% NP-40, 20 mM Tris-HCl (pH 7.4), and 100 mM NaCl, the bound proteins were separated by SDS-PAGE and were subjected to immunoblot analysis.

Immunohistochemistry

Breast cancer tissues were fixed with 4% paraformaldehyde in sodium phosphate (NaPi) buffer (pH 7.4) for 6 h, and then incubated in NaPi buffer containing 30% sucrose for 12 h. The Histofine Simple Stain Kit (Nichirei) for rabbit IgG was used to immunostain frozen 8 µm-tissue sections with anti-Aurora-A or anti-phosphorylated-Aurora-A (T288P) antibodies. Endogenous peroxidase and biotin were blocked, and sections were incubated for 3 h at room temperature with a 1 : 1000 dilution of antibodies to Aurora-A or for 20 h at 4 °C with a 1 : 500 dilution of antibodies to phosphorylated-Aurora-A (T288P). Specimens were then incubated with streptoavidin conjugated anti-rabbit IgG antibodies. After blocking endogenous peroxidases, streptoavidin were labeled with biotin-conjugated horseradish peroxidase, which were detected by diaminobenzidine tetrahydrochloride. Nuclei were counterstained with hematoxylin.

Northern blots

FirstChoiceTM Human Blot 2 (Ambion) were probed with a full length of h-CPEB cDNA that was radioactively labeled by using a random primer (TaKaRa) and [{gamma}-32P]dCTP (Amersham). An overnight hybridization at 42 °C was followed by washing twice in 0.1% SDS containing 1 x SSPE buffer (1 x SSPE consists of 0.18 M NaCl, 10 mM NaH2PO4 and 1 mM EDTA [pH 7.7]) for 15 min at 60 °C and twice in 0.1x SSPE-0.1% SDS buffer for 30 min at 60 °C, following autoradiograph analysis.

Poly(A) tail length (PAT) assay

Total RNA was isolated using RNeasy Mini Kit (Qiagen). The PAT assay was carried out according to Salles et al. with some minor modifications. In brief, 1 µg of total RNA was used for reverse transcription using an anchoring nucleotide-fused oligo-(dT)12 primer (5'-AATGCCAGCTCCGCGGCCGCGTTTTTTTTTTTT-3'). Subsequently, PCR was carried out using an anchoring sequence (5'-GCCAGCTCCGCGGCCGCG-3') and a sense primer targeting a specific sequence in the cDNA of interest (cyclinA2: 5'-CCTCAAAGCACCACAGCATG, cyclinB1: 5'-CTCTTCTTCCAGTTATGCACCACC, cyclinD3: 5'-ATGTAGTCCGTGCTGACAGC, cyclinE1: 5'-CAGAGCGGTAAGAAGCAGAG, Cdk1 : 5'-CCAAACGAATTTCTGGCAAAATGGCACTG, Cdk2: 5'-CAAGGCAGCCCTGGCTCACC, c-myc: 5'-TGAAAAGGCCCCCAAGGTAG, Raf-1: 5'-TTCCATTGAGCTGCTCCAAC, Bcl-2: 5'-GCAAGAGTGACAGTGGATTGC, Survivin: 5'-CATCGAGCAGCTGGCTGCCATG, IAP-1: 5'-AGGAGTACAATCAAGGGTACAG, XIAP: 5'-GTGTCCCATGTGCTACACAGTC, eIF4E: 5'-GCTGTTACACATATAGGGAG, PABPC1: 5'-AGAAAGCAGTTAACAGTGCC, CaMKII: 5'-TCATCCGCATCACGCAGTAC, ß-actin: 5'-AGCAGGAGTATGACGAGTCC). The PCR products were then resolved in 3% agarose gel and visualized by ethidium bromide staining.


   Supplementary Material
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
 Supplementary Material
References
 
The following supplementary material is available for this article online:

Figure S1 Cell cycle analysis for Aurora-A and/or h-CPEB expressing cells. DNA contents of logarithmically proliferating Rat1 cells that stably express wild-type Aurora-A and/or h-CPEB or kinase inactive Aurora-A were analyzed by flow cytometry. To prepare samples for flow-cytometric analysis, 5 x 105 of Rat1 cells were fixed with ethanol and were stained for DNA by incubating in phosphate-buffered saline containing 100 µg/mL propidium iodide and 40 U/mL of RNase A for 30 min at 37 °C. Samples were analyzed by the fluorescence-activated cell sorter (FACS) using the CellQuest software (Beckton Dickinson). The percentages of cells in G1 phase (M1) and in G2/M phase (M2) are shown.

Figure S2 (A) Synergistic elongation of poly(A) by Aurora-A and CPEB in Rat1 cells. Poly(A) tail extension in cells with exogenous expression of wild-type Aurora-A (WT) and/or h-CPEB, or kinase-inactive Aurora-A (KD) and PAT assay was performed as in Fig. 5A. Similarly to the experiment in MCF7 cells, exogenous expression of CPEB in addition to Aurora-A (WT) shows synergistic effect on the poly(A) elongation for Cdk1 and cyclin B1, but not for cyclin A2, survivin and Bcl-2. (B) Additional results of PAT assay in MCF-7 cells. MCF-7 cells were transfected with the indicated combination of plasmid DNA encoding Flag-h-CPEB, HA-Aurora-A(WT), HA-Aurora-A(KD), and Myc-Ajuba and PAT assay was performed after RNA extraction, as in Fig. 6B. No significant change in poly(A) tail length were observed for c-myc, Raf-1, survivin, eIF4E, PABCP1, CaMKII mRNA.


   Acknowledgements
 
We are grateful to Drs T. Iwanaga and N. Hirota for valuable suggestions on immunohistochemical analysis; Y. Fukushima in preparation of the manuscript; J.R.A. Hutchins for the critical reading of the manuscript; and members of the Gene Technology Center in Kumamoto University for their technical support. T.H. is supported by a fellowship from the Japan Society for the Promotion of Science (JSPS). This work was supported by the Ministry of Education, Culture, Sports, Science and Technology of Japan and the Research for the Future program of JSPS (H.S.).


   Footnotes
 
Communicated by: Tadashi Yamamoto

aPresent address: Research Institute of Molecular Pathology, Dr Bohr-gasse 7, A-1030, Vienna, Austria Back

* Correspondence: E-mail: hirota{at}imp.univie.ac.at


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 Introduction
 Results
 Discussion
 Experimental procedures
 Supplementary Material
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Received: 4 March 2005
Accepted: 30 March 2005




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