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1 State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, Shanghai 200433, P. R. China
2 Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA
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Abstract |
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Introduction |
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The Aurora kinase family, belonging to serine/threonine protein kinases, is currently one of the most intensely studied families that regulate many processes during cell division (reviewed in Nigg 2001; Andrews et al. 2003; Carmena & Earnshaw 2003). In mammalian cells, it comprises Aurora A, B and C. Aurora A is localized at the spindle poles from prophase to telophase and has emerged as a critical factor in the assembly of the mitotic spindle. Depletion of Aurora A by RNAi (RNA interference) disrupts centrosomal recruitment of D-TACC (Drosophila transforming-acidic-coiled-coil protein) (Giet et al. 2002), centrosomin in D. melanogaster (Terada et al. 2003) and 
-tubulin in S. cerevisiae (Hannak et al. 2001), leading to defects in microtubule and centrosome morphology. Over-expression of Aurora A causes an increase in centrosome number and aneuploidy, leading to the transformation of mammalian cells (Bischoff et al. 1998). Aurora B is a chromosomal passenger protein, localized on centromeres from prophase to the metaphase-anaphase transition. It then transfers to spindle midzone in anaphase and the cortex where the contractile ring subsequently forms. At last it is associated with the midbody during cytokinesis. Histone H3, an excellent substrate for Aurora B, is phosphorylated at both Ser 10 and Ser 28 in mammals (Goto et al. 2002). In D. melanogaster, H3 fails to be phosphorylated during mitosis after Aurora B was deleted by RNAi and both chromosome condensation and segregation are defective (Giet & Glover 2001). In C. elegance, disruption of Aurora B by RNAi leads to polyploidy (Schumacher et al. 1998). Many lines of evidences show that Aurora B is associated with other two passenger proteins, Survivin and INCENP (inner centromere protein). Disruption of any one of these three proteins interferes with the localizations of the other two, indicating that their accurate targetings and functions during mitosis require each other (Honda et al. 2003). Recently Borealin has been reported as a new part of the vertebrate chromosomal passenger complex containing Aurora B, INCENP and Survivin (Gassmann et al. 2004; Sampath et al. 2004), which suggests this large complex may include other new members. Intriguingly, each of Aurora B, Survivin and INCENP is detected to be over-expressed in many cancer cell lines. These facts lead to the idea that the coordinated increases in the complex and the enhanced Aurora B activity through increased expression of Survivin, are important to transformation or tumor formation (reviewed in Crane et al. 2004).
Aurora C, highly expressed in mammalian testis (Tseng et al. 1998; Bernard et al. 1998; Kimura et al. 1999), was first identified in a screen for kinases expressed in mouse sperm and eggs (Tseng et al. 1998). Its gene maps to 19q13.43, a region often translocated or deleted in certain cancer tissues (Bernard et al. 1998; Kimura et al. 1999) and its protein product is also over-expressed in certain cancer cell lines and in primary colorectal cancers (Kimura et al. 1999; Takahashi et al. 2000). Aurora C protein level is low during S phase and peaks at mitosis (Kimura et al. 1999). Just as mentioned above, recent study has shown that human Aurora C, as a novel chromosomal passenger protein, may regulate mitotic chromosome dynamics in accordance with Aurora B in human cells (Li et al. 2004). However, the real biological functions of Aurora C remain problems.
Herein, we provide evidence that, first, not only is the mRNA of human Aurora C expressed highly in testis, but it also occurs in all other 16 tested tissues at a low level. Second, Aurora C is co-localized with Aurora B and Survivin as a chromosomal passenger protein. Third, Aurora C can form a complex with Aurora B and Survivin and be directly associated with Survivin but not Aurora B. Fourth, like Aurora B, the kinase activity of Aurora C contributes to its proper localization. Finally, both the catalytically inactive Aurora C and its WT (wild-type) are demonstrated to lead to the multinucleation in cells and the Aurora C WT is able to rescue the multinucleation induced by catalytically inactive Aurora B, vice versa. Taken together, these results lead to the hypothesis that Aurora C possesses a similar function to Aurora B and is essential to cytokinesis.
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Results |
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Testis specific expression of Aurora C had been identified in human and mouse by Northern blotting analyses (Bernard et al. 1998; Tseng et al. 1998; Kimura et al. 1999). Using RT-PCR, a more sensitive method, we have found there were two Aurora C splicing forms that varied only in N-terminal and expressed at the highest level in human testis when the full-length of Aurora C cDNA was amplified (Yan et al. 2005). Here, we amplified the C-terminal of Aurora C to further observe the potential distribution of Aurora C mRNA in human 17 tissues. A 450 bp amplicon of C-terminal of Aurora C was visualized in amplification product from commercially prepared cDNAs from 17 human tissues mRNA (Fig. 1). The result showed that despite the highest level of Aurora C mRNA in testis, it was also expressed at a low level in all other 16 tissues.
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The method of cells transfection with vector expressing EGFP (enhanced green fluorescent protein) fusion protein is often used to observe the subcellular localization of the fusion protein. To determine whether the localization of EGFP-tagged Aurora B in HeLa cells is the same as that by immunostaining the endogenous Aurora B, HeLa cells were first synchronized by double treating with thymidine so as to get more G2/M phase cells, then fixed and immunostained by monoclonal antibody against rat Aurora B (AIM-1) which can also recognize human Aurora B. From the up panel of Fig. 2A, we found that during mitosis, Aurora B had a chromosomal localization from prophase to metaphase, transferred to the spindle midzone when the sister chromatids started to separate. After anaphase, it subsequently re-located to the cortex where the contractile ring formed. During cytokinesis, it looked like two little proximate dots telescoped between the two daughter cells.
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To obtain the accurate localization of Aurora C in the processes of mitosis, we transiently expressed Aurora C-EGFP fusion protein in HeLa cells. Consistent with recent discovery (Li et al. 2004), Aurora C was also found to be a chromosomal passenger protein that locates to the same positions as Aurora B in cell mitosis (Yan et al. 2005). Since the subcellular distribution of Aurora C is in accordance with Aurora B, could these two kinases be co-localized? To prove this, HeLa cells that expressed Aurora C-EGFP were fixed and directly stained with monoclonal antibody against Aurora B. As expected, from prophase to cytokinesis, Aurora C-EGFP and Aurora B were co-localized excellently (Fig. 2B).
Survivin is also a chromosomal passenger protein that has the same distribution as Aurora B and is required for targeting Aurora B (Chen et al. 2003). Aurora B kinase is not localized to the kinetochores in fission yeast or C. elegance embryos lacking Survivin (Speliotes et al. 2000; Morishita et al. 2001). After validating the co-localization of Aurora B with Aurora C, we speculated Survivin could also be co-localized with Aurora C. HeLa cells containing Aurora C-EGFP were stained with antibody against human Survivin and observed by fluorescence microscopy. As shown in the merged photos of Fig. 2C, we confirmed the co-localization of Aurora C-EGFP and Survivin during mitosis.
Thus, these findings demonstrated that it is most likely that Aurora C is associated with Aurora B and Survivin as a chromosomal passenger protein. Perhaps, through directly binding to Aurora B and/or Survivin, Aurora C forms into a complex with Survivin and Aurora B. At this rate, Aurora C might catalyze the same substrates and exert the same functions as Aurora B.
Aurora C is associated with Aurora B and Survivin in vivo and directly binds to Survivin in vitro
To determine whether Aurora C is associated with Aurora B and Survivin in cells, we produced transiently transfected HEK 293T cells that expressed HA (hemagglutinin)-tag alone or HA-tagged Aurora C. The cell lysates were immunoprecipitated with monoclonal antibody against Aurora B and polyclonal antibody against Survivin, respectively. We subsequently immunobloted the immunoprecipitates with the anti-HA, anti-Survivin and anti-Aurora B antibody, respectively. As shown in Fig. 3A, lane 4, the complex pulled down by anti-Aurora B antibody contained both HA-tagged Aurora C and endogenous Survivin. Consistent with this, the complex pulled down by anti-Survivin antibody also contained both HA-tagged Aurora C and endogenous Aurora B (Fig. 3A, lane 6).
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Previous studies have shown that Aurora B interacts directly with Survivin in both two-hybrid and in vitro pull down assays, in which the complex is stable in treatments as harsh as 3 M NaCl (Wheatley et al. 2001). Survivin can specifically bind to the catalytic domain of Aurora B (Chen et al. 2003). In view of these, we ulteriorly investigated whether Aurora C also directly binds to Survivin or Aurora B. In vitro GST (glutathione S-transferase)-pull down assays, using glutathione agarose beads to incubate together with GST-tagged Survivin and His-tagged Aurora B, confirmed the result that Aurora B interacted directly with Survivin (Fig. 3D). In the same way, we revealed Aurora C could also directly bind to Survivin (Fig. 4E). However, no matter whether GST-Aurora C/His-Aurora B or GST-Aurora B/His-Aurora C was used, we did not detect direct interaction between Aurora B and Aurora C (Fig. 3D,E).
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Improper localization of the catalytically inactive Aurora C
As suggested in recent studies, the catalytically inactive Aurora B K106R mutant failed to locate properly and seemed to distribute diffusely to either centromeres or the spindle midzone, which severely impaired mitotic progression (Honda et al. 2003). After the discovery of the association of Aurora C with Aurora B, the localization of kinase inactive Aurora C in mitosis was therefore of great interest. We produced the kinase inactive Aurora C D166Y mutant by site mutation (Fig. 4A). Through transfecting Aurora C D166Y-EGFP and Aurora B K106R-EGFP into HeLa cells, we found that the kinase inactive Aurora C, like kinase inactive Aurora B, could still be localized to chromosomes from prophase to metaphase (Fig. 4B). However, after the chromosomes separation, both kinase inactive Aurora C and Aurora B were not localized to the spindle midzone and then midbody as usual, but remained with the chromosomes until the end of mitosis. Eventually, without the help of general kinase activity and accurate localization of Aurora B or C, many cells could not accomplish cytokinesis, resulting in two nuclei in one cell (Fig. 4B). These findings gave us an indication that the kinase activity of Aurora C, as well as Aurora B, is required for its transfer from DNA to the spindle midzone. Without the kinase activity, neither Aurora C nor Aurora B is localized properly with the progression of mitosis.
However, in some cells expressing only low amounts of Aurora C or Aurora B mutant, due to the slight dominant-negative effects, these inactive proteins were localized both in the cortex or midbody and in DNA from telophase to cytokinesis. Moreover, in some cells undergoing cytokinesis, they remained in midbody as usual, only showing irregular shapes (Fig. 4C). All these cells showed no evidence of mitotic aberration.
Over-expression of kinase inactive Aurora C or Aurora B lead to multinucleation, which can be rescued by WT of Aurora B or Aurora C, respectively
Since the over-expression of Aurora B K106R mutant or WT was reported to disrupt cleavage furrow formation without affecting nuclear division, leading to multinucleate cells (Tatsuka et al. 1998), we tested whether the over-expression of Aurora C D166Y mutant or WT frequently induces multinucleation. Comparing the effects of over-expressing D166Y mutant/WT of Aurora C-EGFP with K106R mutant/WT of Aurora B-EGFP in HeLa cells, we found cells positively expressing the EGFP with Aurora C mutant had a dominant-negative effect of inducing multinucleation at 72 h after transfecion. WT of Aurora C, on the other hand, induced a similar phenotype at a much lower level after 72 h induction. Meanwhile, the mutant or WT of Aurora B also separately caused multinucleation, the percentages of which were higher than those induced by the mutant or WT of Aurora C, respectively (Fig. 5A). We also co-transfected Aurora B mutant together with Aurora C mutant into HeLa cells. Due to the coeffects of two kinase inactive mutants, cells induced much greater multinucleation. This also occurred in WT of Aurora B and Aurora C (Fig. 5A). These findings illustrated that the function of Aurora C in cytokinesis might be similar to Aurora B, owing to their parallel expression-mediated phenotypic effects.
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Discussion |
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Aurora C has been found to be expressed highly and specifically in mammalian testis by Northern blot analyses (Bernard et al. 1998; Tseng et al. 1998; Kimura et al. 1999). However, using the more sensitive method of RT-PCR, we found Aurora C mRNA exists in all 17 tested human tissues including testis, which still contain the largest amount of Aurora C. This means Aurora C may be indispensable to the basic cell proliferation in all tissues. The expression of human Aurora A and B is also high in testis. As it is known that mature testis continuously process meiosis and mitosis, it might require abundant Aurora A, B and C to ensure continuous production of spermatogonia, spermatocytes, spermatoblasts and sperms.
By applying both anti-Aurora B monoclonal antibody and Aurora B-EGFP vector to locate Aurora B in HeLa cells, we achieved the same result that Aurora B is a chromosomal passenger protein. Therefore, the selective targeting of chromosomal passenger protein was not affected by the C-terminal EGFP-tag in transfected system. Without a specific antibody against Aurora C, we adopted the technique of tracing EGFP-fusion Aurora C in HeLa cells. In our results, the behavior of Aurora C is the same as Aurora B. We assumed that Aurora C is co-localized with Aurora B and other chromosomal passenger proteins such as Survivin. As expected, these three proteins occur in the same positions during cell division, which gave us an indication that Aurora C may be associated with Aurora B and Survivin in a complex.
Survivin, a member of the inhibitor of apoptosis (IAP) gene family (Deveraux & Reed 1999), is expressed in mitosis in a cell cycle-dependent manner and is localized to the mitotic apparatus (Li et al. 1998). Interference with the expression/function of endogenous survivin resulted in increased caspase-3 activity at G2/M (Li et al. 1998) and a profound dysregulation of mitotic progression (Li et al. 1999), with supernumerary centrosomes, aberrant mitotic spindles, polyploidy and lethal defects of cytokinesis. The structure of human survivin forms a very unusual bow tie-shaped dimer with two 
-helical extensions (Chantalat et al. 2000). The C-terminal of survivin is reported to be required for binding to microtubules in vitro. The shape of the survivin dimer permits a unique adaptor or docking function on the microtubule polymer or elsewhere. In the chromosomal passenger protein complex, Survivin directly interacts with both the C-terminal of Aurora B and the N-terminal of INCENP (Wheatley et al. 2001; Bolton et al. 2002; Chen et al. 2003). INCENP is another important member of the chromosomal passenger complex that is essential to cell division. RNAi mediated down-regulation of INCENP in elegans and Drosophila produced severe mitotic defects (Kaitna et al. 2000; Adams et al. 2001). Similar defects were also seen on over-expression of a dominant-negative INCENP mutant in vertebrate cells (Mackay et al. 1998). Of particular interest, all metazoan INCENPs share a highly conserved motif near the C-terminus, the so-called IN-box (Bolton et al. 2002). In Xenopus, one mapping data is consistent with a scaffold protein role for INCENP, which binds to Survivin on its N-terminal and Aurora B on its IN-box of C-terminal (Bolton et al. 2002). Aurora B phosphorylates the IN-box primarily on the three residues making up the TSS motif and the phosphorylation of TSS enhances the ability of INCENP to strongly activate Aurora B in a feedback way (Honda et al. 2003). The data on the structural relationships among the members of the chromosomal passenger complex shows that any one of Aurora B, Survivin and INCENP binds to the other two.
Using a series of experiments of immunoprecipitation, we further presented evidence that in vivo Aurora C is part of the complex containing Aurora B and Survivin. By NCBI-BLASTp (http://www.ncbi.nlm.nih.gov/blast/bl2seq/bL2.html) we found that the full length of Aurora C has 76% identities and 85% positives with the full length of Aurora B, but the catalytic domain of these two kinases have 84% identities and 91% positives, which gave the possibility that their catalytic domain could have similar structures. Therefore, it is possible that besides Aurora B, Survivin could also bind to the catalytic domain of Aurora C. Through in vitro GST-pull down assays, we proved that Survivin binds to Aurora C. However, we did not find that Aurora C interacts directly with Aurora B. Recently Li et al. (2004) have reported that Aurora C is also associated with INCENP and directly binds to IN-box of INCENP in vivo, which activates Aurora C. Therefore, colligating the results of Li et al. (2004) and us, we speculated that by the direct binding to Survivin and/or the IN-box of INCENP, Aurora C and Aurora B were able to be associated together within the same complex. Certainly, more experiments are needed to clarify the detailed structural relationship among the members of the large and complicated complex.
By mutant analysis, our results suggested that Aurora C kinase activity, like Aurora B, contributes to its accurate localizations. Even though, in the majority of the mitotic cells, the catalytically inactive Aurora C and B could still be present in chromosomes before the sister chromatids separation, the inactive proteins seemed to be diffuse along all the chromosomal arms. In the subsequent mitotic events, both the inactive Aurora C and B were not efficiently localized to the right positions. The most straightforward conclusion from these results is that the phenotype of Aurora C mutant is in line with that of Aurora B mutant.
Expression of the kinase inactive Aurora C exerted a dominant-negative effect and resulted in a terminal phenotype characterized by multinucleation, which confirmed the notion that Aurora C is also critical for cytokinesis, in agreement with previous findings of Aurora B (Honda et al. 2003). The molecular mechanisms leading to multinucleation remain to be completely understood. However, whatever inactive kinase or WT was over-expressed in cells, the effect of Aurora C was less than that of Aurora B. This raised the possibility that the abundance or the kinase activity of Aurora C is lower than Aurora B in mitotic cells. When the kinase inactive Aurora C was expressed in cells, it could be complemented by endogenous kinase active Aurora B and vice versa. But when these two inactive kinases were expressed together, this kind of complement did not occur being replaced by severer multinucleation.
Finally, the mutual rescue abilities of Aurora B and C deeply illuminated that the function of Aurora C overlaps with that of Aurora B during cytokinesis. Aurora C, a novel chromosomal passenger protein, might cooperate with Aurora B to play an important role in regulating mitosis. Nevertheless, whether there are functional differences between Aurora C and B and whether Aurora C possesses the distinctive characters crucial to mitosis and meiosis remain to be investigated in future.
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Experimental procedures |
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Human MTC (multiple-tissue cDNA, Clontech, Palo Alto, CA, USA) panel, including bone marrow, stomach, bladder, lung, placenta, pancreas, heart, spleen, liver, thymus, testis, colon, uterus, ovary, brain, skeleton muscle, prostate, is served as the template to study the distribution of human Aurora C mRNA. Aurora C sense primer (5'-GTATAACTATTTCCATGATGCACGC-3') and anti-sense primer (5'-CTCACATCTACCTTGAGGATGCGT-3') were used to amplify a 450 bp fraction of the C-terminal of Aurora C cDNA. PCR was performed in a 25 µL solution at 94 °C (40 s), 53 °C (40 s) and 72 °C (30 s) for 32 cycles. ß-MG (ß-macroglobe) sense primer (5'-ATGAGTATGCCTGCCGTGTGAAC-3') and anti-sense primer (5'-TGTGGAGCAACCTGCTCAGATAC-3') were used to amplify a 290 bp amplicon of C-terminal of ß-MG, which is served as the internal control. PCR was performed in the 25 µL solution at 94 °C (30 s), 60 °C (30 s) and 72 °C (30 s) for 30 cycles. These products were analyzed by electrophoresis in a 1% (w/v) agarose gel stained with ethidium bromide.
Site-directed mutagenesis and plasmids construction
Human Aurora B (GENBANK accession no. AF008552), Aurora C (GENBANK accession no. AF054621) and Survivin (GENBANK accession no. BC034148) were cloned from a human testis cDNA library (Clontech). The D166Y mutant of Aurora C and K106R of Aurora B were obtained by site-directed mutagenesis using the QuikChange® site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA) according to manufacturer's instructions. To investigate subcellular localization, the cDNAs of Aurora C WT and D166Y mutant, Aurora B WT and K106R mutant were individually introduced into the pEGFP-N1 vector (Clontech). The EGFP sequence is located at the 3' end of the kinase sequence. For the mammalian expression of the proteins, the Aurora B, Aurora C and Survivin cDNAs were inserted into pCMV-myc or pCMV-HA vecors (Clontech). For the bacterial expression of the fusion proteins, these cDNAs were subcloned in frame with GST tag into pGEX4T-1 vector (Amersham Biosciences, Buckinghamshire, UK) and His tag into pET32a(+) vector (Amersham Biosciences).
Cell culture, synchronization and transfection
HEK 293T and HeLa cells were cultivated in Dulbecco's modified Eagle's medium (Gibco, Carlsbad, CA, USA) supplemented with 10% (v/v) foetal calf serum (Invitrogen, Carlsbad, CA, USA) at 37 °C in 5% CO2-humidified atmosphere. Exponentially growing HeLa cells were blocked for 22 h with thymidine (final concentration 2 mM), washed three times with PBS (phosphate-buffered saline) then incubated with fresh medium. After 12 h, thymidine was added again and the cells were incubated for an additional 22 h. Plates were then washed and added with fresh medium. After 10 h (at this moment the majority of cells is in G2/M phase by the FACS analysis), cells were collected for the immunostaining of endogenous Aurora B. For cell transfection, HEK 293T or HeLa cells with 80% confluent were transfected with plasmids using LipofectamineTM Reagent (Invitrogen) in the non-serum medium. After 5 h of incubation, medium was replaced with fresh complete medium and cells were harvested at different times before being analyzed.
Immunofluorescence microscopy
Cells grown on coverslips were fixed in preiced 3.7% (w/v) formaldehyde/PBS (pH 7.0) for 10 min. After being washed with PBS 3 times, cells were resolved by 0.2% (v/v) Triton X-100 for 15 min at room temperature. and then the permeabilized cells were blocked by a solution containing 10% (v/v) horse serum and 1% (w/v) BSA in PBS for 1 h at room temperature. The diluted monoclonal antibody against rat Aurora B (anti-AIM1, BD Biosciencs, Palo Alto, CA, USA) or goat polyclonal antibody against Survivin (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was placed as a drop on the coverslips and incubated for 2 h at 37 °C in a humidified chamber. The cells were then washed and covered with Rhodamine-conjugated anti-mouse or Cy3-conjugated anti-goat antibody, respectively, for 1 h in the dark. At last, the cells were counterstained with DAPI (1 µg/µL 4, 6-diamidino-2-phenylindole) at 37 °C for 20 min. Images were acquired using a LEICA DC 500 camera on a microscope equipped with LEICA DMRA2 fluorescent optics (LEICA, Germany).
Immunoprecipitation
HEK 293T cells in 60 mm dishes were lyzed in 1 mL of cell lysis buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% (v/v) Triton X-100, 2.5 mM sodium pyrophosphate, 1 mMß-glycerophosphate, 1 mM Na3VO4, 1 µg/mL leupeptin) for 30 min at 4 °C on a rotating wheel. The lysates were centrifuged at 15 000 g for 15 min and the supernatants were preclarified by incubating with protein A/G-Sepharose beads (Oncogene, San Diego, CA, USA) for 1 h. After the beads were discarded by centrifugation, the lysates were then immunoprecipitated with primary antibody bound to protein A/G-Sepharose beads for 6 h at 4 °C. The beads were washed five times with 1 mL of cell lysis buffer, boiled in 2x SDS sample buffer and finally analyzed by Western blotting.
Purification of recombinant protein
The recombinant GST or His tagged proteins were expressed in Escherichia coli strain BL21-CodonPlus(DE3)-RIL with 0.5 mM IPTG (isopropyl-ß-D-thiogalactopyranoside) at 25 °C after 3 h or overnight induction, respectively. Cells were collected and lyzed in the buffer of PBS (pH 7.0) or the buffer containing 50 mM NaH2PO4 (pH 8.0), 300 mM NaCl, 10 mM imidazole, respectively, supplemented with 1% (v/v) Triton X-100, 10 mMß-mercaptoethanol, 0.5 mM PMSF and 1 mg/mL lysozyme. After incubation on ice for 30 min, the samples were sonicated and centrifuged. The supernatants were purified using Glutathione Sepharose 4B (Amersham Biosciences) or Ni-NTA His-bind® Resins (Novagen, Madison, WI, USA), respectively, as recommended by the manufacturers.
In vitro binding assay
For each binding reaction, 10 µg of purified His-Aurora C (His-Aurora B) was added into binding buffer (PBS, 5 mM EGTA, 0.1% (v/v) Triton X-100, 0.5 mM PMSF) containing 8 µg of GST alone or 5 µg of GST-Survivin or 15 µg of GST-Aurora B (GST-Aurora C) prebound to glutathione agarose beads. Samples were incubated on a rotating wheel at 4 °C for 2 h. The beads were washed 5 times with binding buffer, boiled in 2x SDS sample buffer and analyzed by Western blotting.
Western blotting
Samples were separated by 12% (v/v) SDS/PAGE and transferred to nitrocellulose membranes, and then these were separately incubated with antibodies against the Aurora B (monoclonal anti-AIM1, BD Biosciences), Survivin (goat polyclonal antibody, Santa Cruz Biotechnology), HA-tag (monoclonal antibody, COVANCE, Berkeley, CA, USA), myc-tag (c-myc monoclonal antibody, BD Biosciences), His-tag (monoclonal anti-polyHistidine antibody, R & D systems, Minneapolis, MN, USA) or GST-tag (GST-tag monoclonal antibody, Novagen), followed by horseradish peroxidase-conjugated rabbit anti-mouse or anti-goat antibodies, respectively. Immunoreactivity was visualized by enhanced chemiluminescence.
In vitro kinase assay
Reaction mixture (20 µL) containing His-Aurora C or its D166Y mutant, 2 µg of MBP (myelin basic protein) as the substrate, 5 µM ATP, 10 µCi of [-32P] ATP in kinase buffer (500 mM HEPES, pH 7.4, 10 mM MgCl2, 10 mM MnCl2, 1 mM EGTA, 1 mM DTT, 5 mMß-glycerophosphate) was incubated at 30 °C for 30 min. Samples were separated by 12% (v/v) SDS/PAGE and visualized using Coomassie Brilliant Blue staining. The gels were dried and subjected to autoradiography.
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Acknowledgements |
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Footnotes |
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* Correspondence: E-mail: longyu{at}fudan.edu.cn
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References |
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Received: 9 February 2005
Accepted: 21 March 2005
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