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Human Molecular Genetics Advance Access originally published online on October 23, 2006
Human Molecular Genetics 2006 15(22):3343-3350; doi:10.1093/hmg/ddl410
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© The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

The N-terminal domain of the Aurora-A Phe-31 variant encodes an E3 ubiquitin ligase and mediates ubiquitination of I{kappa}B{alpha}

Paraskevi Briassouli1,{dagger}, Florence Chan2 and Spiros Linardopoulos1,2,*

1 The Breakthrough Breast Cancer Research Centre, The Institute of Cancer Research, Fulham Road, London SW3 6JB, UK and 2 Cancer Research UK Centre for Cancer Therapeutics, The Institute of Cancer Research, Haddow's Laboratories, 15 Cotswold Road, Sutton SM2 5NG, UK

* To whom correspondence should be addressed. Tel: +44 2071435341; Fax: +44 2071435340; Email: spiros.linardopoulos{at}icr.ac.uk

Received August 30, 2006; Revised September 25, 2006; Accepted October 6, 2006


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Aurora-A is an important regulator of mitosis and is frequently amplified in human cancer. Ectopic expression of Aurora-A in mammalian cells induces centrosome amplification, genomic instability and transformation. A common genetic variant in Aurora-A (F31I) is preferentially amplified and is associated with the occurrence and the status of colon, oesophageal and breast cancers. Here we demonstrate that the N-terminal domain of Aurora-A Phe-31 variant exhibits an intrinsic ubiquitin ligase activity. Mutation of cysteines 8, 33 and 49 of Aurora-A abolishes the ubiquitin ligase activity of the protein. Aurora-A in a complex with UBE2N/MMS2 catalyses polyubiquitination of I{kappa}B{alpha} in vitro and in vivo.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The Aurora-A gene encodes a cell-cycle-regulated serine/threonine kinase (1). Aurora-A is the human homologue of the aurora protein kinase from Drosophila, Ipl1 kinase from Saccharomyces cerevisiae (2,3), and is a member of a family of Aurora kinases that includes Aurora-B and Aurora-C. The Aurora-A gene is located in the 20q13 chromosome region that is frequently amplified in a variety of malignant tumours such as colorectal, breast and bladder cancers (4,5). However, its upstream regulators and downstream targets are poorly understood and characterized.

Ectopic expression of Aurora-A in Rat1 and NIH3T3 cells causes centrosome amplification, chromosome instability, transformation in vitro and tumourigenesis in nude mice (4–6). However, overexpression of Aurora-A in primary MEF cells does not induce transformation indicating that Aurora-A alone is insufficient to induce carcinogenesis (7). In agreement with this, a transgenic mouse designed to overexpress Aurora-A in mammary epithelium did not develop tumours (8) indicating that additional factors in association with Aurora-A promote tumourigenesis.

A polymorphism in the Aurora-A gene, Phe31Ile, has recently been identified (5). The 91A>T causes Ile>Phe substitution at amino acid position 31. The heterozygous state of the polymorphism (Phe31Ile) is ~30%, whereas the homozygous state of the Ile31Ile allele is ~4% in Caucasian population. This polymorphism is located the within an N-terminal conserved motif, Aurora Box 1 (amino acids 5–40), which is hypothesized to be involved in ubiquitin-dependent destruction (9,10). In particular, the amplification of the Ile-31 variant has recently been demonstrated to be associated with increased aneuploidy in colon cancers. Even though both variants had transforming properties in rodent cells, the Ile-31 variant showed stronger transforming activity than the Phe-31 (11). The Aurora-A Ile-31 isoform was also associated with an increased risk of oesophageal squamous cell carcinoma, breast cancer and ovarian cancer compared with the Aurora-A Phe-31 isoform (12–14). Moreover, in case–control studies involving colon, breast, ovarian, prostate, lung, oesophageal and non-melanoma skin cancer, it was confirmed that the Aurora-A Ile-31 variant is a low penetrance cancer susceptibility allele affecting multiple cancer types (15). These findings suggest that Aurora-A is a crucial kinase and its functional polymorphisms are susceptibility variants in various human tumour types.

We have recently identified Aurora-A Phe-31 as a binding partner of E2 ubiquitin ligase UBE2N, the human homologue of Ubc13, in vitro and in vivo (11). UBE2N has been shown to be involved in NF-{kappa}B activation via I{kappa}B{alpha} degradation (16). The function of the non-catalytic N-terminus of Aurora-A is poorly understood, although it contains binding sites for several proteins (5). On the basis of these observations we aimed to investigate the biochemical properties of the Aurora-A Phe-31 variant in association with the E2 ligase UBE2N. We found that the N-terminal domain of Aurora-A Phe-31 variant exhibited intrinsic ubiquitin ligase activity. Aurora-A in a complex with the E2 ubiqitin ligase UBE2N/MMS2 catalysed I{kappa}B{alpha} polyubiquitination in vitro and in vivo. The significance of the association between Aurora-A Phe-31 and I{kappa}B{alpha} and its role in tumourigenesis needs to be further investigated.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
UBE2N ubiquitinates Aurora-A
Recently, linkage analysis and haplotype mapping in mice identified Aurora-A as a candidate skin tumour susceptibility gene (11). In humans association studies have revealed that Aurora-A presents polymorphisms among the population (5). One such polymorphism (Ile-31) was elevated in tumours and amplification of the allele carrying this change leads to increased cancer risk (11). Furthermore, we have also identified that the Aurora-A Phe-31 variant showed higher affinity for the E2 ligase UBE2N in vitro and in vivo (11). UBE2N is involved in proteasome-independent Lys-63 ubiquitin chain assembly (16). Characterization of human tumour cell lines by Taqman real-time PCR assay showed that HeLa and A549 cells are homozygous Phe-31 or Ile-31, respectively (data not shown). To determine the effect of UBE2N on Aurora-A Phe-31 and Ile-31 variants, HeLa and A549 cells were transfected with increasing amounts of wild-type UBE2N and a constitutively inactive mutant UBE2N-C87A (16). UBE2N overexpression induced ubiquitination and stabilization of Aurora-A in HeLa but not in A549 cells (Fig. 1A). To characterize the ubiquitin ligase activity of UBE2N/MMS2 complex towards Aurora-A, a His-Aurora-A fusion protein containing residues 1–117 of Aurora-A Phe-31 variant, was incubated with ATP, E1, 1 µg of HeLa extracts (as E3), and ubiquitin. UbK48R mutant ubiquitin was also used to determine the type of ubiquitin linkage formed. The continued ubiquitination of Aurora-A (amino acid 1–117) in the presence of UbK48R indicates that UBE2N promotes Lys48-independent ubiquitin chain assembly (Fig. 1B).


Figure 4101
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  		Figure 1. Aurora-A ubiquitination by UBE2N. (A) HeLa and A549 cell lines were transfected with increased amount of MYC-tagged UBE2N or HA-tagged UBE2N(C87A) mutant and cell lysates were subjected to western blot using anti Aurora-A and UBE2N levels were assessed using anti-MYC- and anti-HA-specific antibodies. (B) N-terminal domain of Aurora-A Phe-31 variant (amino acid 1–117) was incubated in the presence of ATP, E1 enzyme, ubiquitin or ubiquitin K48R and HeLa lysates (1 µg) as E3 with UBE2N. Ubiquitinated Aurora-A was assessed by western blot with anti-IAK1 antibody.

 
Aurora-A exhibits E3 ligase activity
The interaction of UBE2N with Aurora-A (11), prompted us to investigate the physiological role of this complex. First, we tested if Aurora ubiquitination by UBE2N/MMS2 complex affects its kinase activity similar to TAK1 kinases (16). We showed that the E2-ligase complex had no significant effect on Aurora-A kinase activity when myelin basic protein (MBP) was utilized as a substrate (data not shown) although, we do not exclude activation of Aurora-A by UBE2N in vivo. E3 ligases are binding partners of E2 enzymes which until recently have been classified into two major families—the HECT domain proteins and the RING finger proteins (17). However, a variety of proteins harbouring domains such as the U-box (18), CUE motif (19) and PHD (20) domain have now been shown to harbour E3 ligase activity. In the case of MEKK1 Ser/Thr kinase, the PHD domain of the kinase has been shown to be responsible for mediating ubiquitination of ERK1 (21). Moreover, proteins with no distinct motif at all, such as p300 have also been shown to function as E3/E4 ligases (22). To determine if Aurora-A shares E3-like ubiquitin ligase activity, we used Aurora-A purified from E. coli, which does not contain ubiquitination enzymes, in an in vitro ubiquitination assay. Recombinant Aurora-A protein mediated polyubiquitination of monomeric ubiquitins (Fig. 2A). Moreover, this Aurora-A ligase activity was shown to be dose-dependent. In response to increasing Aurora-A levels, an increase in polyubiquitin chain formation was observed (Fig. 2B). This enzymatic activity of Aurora-A was E2-dependent since in the absence of E2 no ubiquitination could be detected. The specificity of Aurora-A for E2s was further evaluated. Various ubiquitin-conjugating enzymes were tested for their ability to cooperate with Aurora-A. Only UBE2N/MMS2 showed efficient ubiquitin-conjugation (Fig. 2C). Thus, in vitro Aurora-A has preference for UBE2N/MMS2. Methylated ubiquitin was used to attempt to abolish polyubiquitin chain formation. As Fig. 2D shows, the presence of methylated ubiquitin abolished the formation of conjugates. Taken together, these data demonstrate that Aurora-A has E3-like ubiquitin ligase activity in vitro. To test the intrinsic nature of the Aurora-A E3 ligase activity in vivo, HeLa cells were transfected with exogenous ubiquitin and equal amounts of Aurora-A variants Phe-31 and Ile-31, Aurora-B, and the catalytic domain of Aurora-A (amino acid 118–403). Only Aurora-A Phe-31 mediated the stabilization of ubiquitin-conjugates suggesting the specificity of this variant to form polyubiquitin chains (Fig. 3A). Similar results were obtained when the ubiquitin K48R mutant was utilized (data not shown). Finally, overexpression of Myc-tagged Aurora-A in HeLa cells led to the appearance of polyubiquitinated Aurora-A Phe-31 but not Ile-31, despite their equal level of expression (Fig. 3B).


Figure 4102
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  		Figure 2. Recombinant Aurora-A is a ubiquitin ligase. (A) Incubation of Aurora-A in the presence of UBE2N/MMS2, E1 enzyme and ubiquitin, led to the formation of polyubiquitin chains detected by blotting with an anti-ubiquitin antibody. (B) Dose-dependent effect of Aurora-A on promoting ubiquitinated conjugates. Increasing amounts of His-tagged Aurora-A were incubated with UBE2N/MMS2, E1 and ATP. Detection of conjugates was done with an anti-ubiquitin antibody. (C) Aurora-A Phe-31 was incubated in the presence of ATP, E1 enzyme, ubiquitin with UBC3, UBC5a, UBC5b, UBC10 or UBE2N recombinant proteins. Anti-ubiquitin specific antibody was used for the detection of ubiquitin conjugates. (D) Aurora-A Phe-31 was incubated in the presence of ATP, E1 enzyme and UBE2N with either ubiquitin or methylated ubiquitin. Anti-ubiquitin specific antibody was used for the detection of ubiquitin conjugates.

 


Figure 4103
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  		Figure 3. (A) Ubiquitin ligase activity of Aurora-A Phe-31. HeLa cells were transfected with MYC-tagged Aurora-A Phe-31, Aurora-A Ile-31, Aurora-B, Aurora-A C-terminal (amino acid 118–403) or empty vector. HA-ubiquitin conjugates were detected by anti-HA-specific antibody. Equal loading was detected by ezrin antibody and levels of transfected proteins were detected by anti-MYC antibody. (B) HeLa cells were transfected with Aurora-A Phe-31 or Aurora-A Ile-31 and 50 µg of lysates subjected to western blotting and detection with Aurora-A specific antibody. (C) Auto-ubiquitination assay of Aurora-A and Aurora-A-cysteine mutants (cysteine 8, 3, 49). Ubiquitination was detected by blotting with an anti-ubiquitin antibody. (D) Alignment of Aurora-A FP CUE-like domain (in bold) with yeast Cue3/Yg110c, Vps9 and Def1 CUE domains. The Aurora-A variant Ile-57 (in bold), it is a conserved residue of CUE domain.

 
During the ubiquitination reaction, E3 ligases form an intermediate thioester bond with ubiquitin, using a cysteine residue. Aurora-A does not possess any canonical E3 ligase domains such as the HECT domain. To search for critical residues on Aurora-A, which could explain its ubiquitin ligase activity, three cysteine residues on the N-terminal domain of Aurora-A were identified at positions 8, 33 and 49, that are absent in both Aurora-B and Aurora-C. To investigate the importance of the cysteine residues in Aurora-A, the C8, C33 and C49 were mutated individually, but no effect on polyubiquitination was observed (data not shown). All three cysteine residues were then mutated and subjected to in vitro ubiquitination assays. As Fig. 3C shows, the Aurora-A protein carrying cysteine mutations were less capable of auto-ubiquitination compared with the wild-type Aurora-A. These data indicate that C8, C33 and C49 of Aurora-A are important for its ligase activity.

Aurora-A binds to and ubiquitinates I{kappa}B{alpha}
Since the Aurora-A Phe-31 variant (i) binds specifically to UBE2N, which participates in the cascade of events leading to I{kappa}B{alpha} ubiquitination (16) and (ii) has ubiquitin ligase activity in addition to its kinase activity, we examined whether Aurora-A directly binds and promotes I{kappa}B{alpha} ubiquitination. First, the interaction of recombinant Aurora-A and I{kappa}B{alpha} was analysed. Recombinant GST-I{kappa}B{alpha} (amino acid 1–54), both wild-type and a S32A/S36A mutant, and control GST-protein were incubated with full length Aurora-A. Aurora-A could directly interact with both wild-type and mutant forms of I{kappa}B{alpha}, indicating that the binding likely occurred in a phosphorylation-independent manner of I{kappa}B{alpha} (Fig. 4A). To investigate whether endogenous Aurora-A binds to endogenous I{kappa}B{alpha}, we immunoprecipitated lysates from MCF7 cells with an anti-Aurora-A antibody followed by immunoblot probing with an I{kappa}B{alpha} antibody (Fig. 4B). This revealed that Aurora-A can be co-immunoprecipitated with I{kappa}B{alpha}, indicating an endogenous interaction between the two proteins. To delineate the region of Aurora-A responsible for its association with I{kappa}B{alpha}, we generated His-tagged N-terminal (amino acid 1–117) and C-terminal (amino acid 118–403) domains of Aurora-A. GST-I{kappa}B{alpha} (amino acid 1–54) fusion protein was incubated with these protein fragments and only the catalytic domain of Aurora-A was involved in interaction with I{kappa}B{alpha} (Fig. 4C and data not shown). Next we examined whether Aurora-A and I{kappa}B{alpha} colocalize in cells. Aurora-A has a characteristic subcellular localization, being associated with the centrosomes during interphase and at the spindle poles during mitosis (23). Indirect immunofluoresence analysis of HeLa cells revealed that I{kappa}B{alpha} colocalizes with Aurora-A at the centrosomes (Fig. 4D).


Figure 4104
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  		Figure 4. In vitro and in vivo interaction between Aurora-A and I{kappa}B{alpha}. (A) HA-tagged recombinant Aurora-A was immobilized on agarose and incubated with GST-tagged wild-type (amino acids 1–54) and mutant (amino acids 1–54, S32A/S36A). The lower panel shows that equal I{kappa}B{alpha} levels were loaded. Recombinant GST and Aurora-A were loaded as controls. (B) Lysates from MCF7 cells were immunoprecipitated with anti-Aurora-A specific antibody and probed with antibody to I{kappa}B{alpha} to detect the binding effect. Pre-immune serum used as control. IP, immunoprecipitation; IB, immunoblot. (C) Recombinant Aurora-A C-Terminal (amino acid 118–403) was incubated with GST-I{kappa}B {alpha}(amino acid 1–54) and glutathione coupled sepharose beads. The proteins were immunoblotted with anti-Aurora-A (top panel). Input controls for I{kappa}B{alpha} and Aurora-A are also shown. Asterisk shows a non-specific band. (D) Confocal microscopy of HeLa cells stained with antibodies for Aurora-A, and phosphorylated-Ser32/36 I{kappa}B{alpha}. Red, Aurora-A; Green, phosphorylated-Ser32/36 I{kappa}B{alpha}; Blue, DNA (a–d). In a control experiment HeLa cells separately stained with antibody for {gamma}-tubulin. Green, {gamma}-tubulin (e) (Bar, 1 cm=25 µ).

 
To test the ability of Aurora-A to ubiquitinate I{kappa}B{alpha}, in vitro translated Aurora-A was incubated with I{kappa}B{alpha} in the presence of UBE2N/MMS2 and the status of I{kappa}B{alpha} modification was assessed. UBE2N promotes a laddering of low molecular weight conjugates of I{kappa}B{alpha} suggesting a low level of ubiquitination (Fig. 5A). Addition of Aurora-A results in a substantial increase of the molecular weight of the modified I{kappa}B{alpha}, demonstrating that Aurora-A may mediate ubiquitination of I{kappa}B{alpha} in vitro. To test whether the ubiquitination of I{kappa}B{alpha} was due specifically to Aurora-A, wild-type Aurora-A, Aurora-A (K162M) kinase inactive or Aurora-B as a control were used in an in vitro ubiquitination assay. Previous studies have shown that UBE2N/MMS2 complex-catalyses the formation of K63-polyubiquitin chains, therefore, recombinant ubiquitin K63-only was added. As Fig. 5B shows, only wild-type Aurora-A led to ubiquitination of I{kappa}B{alpha} suggesting that the kinase activity of Aurora-A may be important for its ubiquitination activity. To test whether Aurora-A promotes ubiquitination of I{kappa}B{alpha} in vivo, MCF7 cells and MCF7 cells overexpressing Aurora-A were used to examine the levels of ubiquitinated I{kappa}B{alpha}. When total I{kappa}B{alpha} was immunoprecipitated from MCF7 and MCF7-Aurora-A cells, an increased amount of ubiquitinated I{kappa}B{alpha} was observed in the MCF7 cells overexpressing Aurora-A as detected using a ubiquitin-specific antibody (Fig. 5C). We conclude that both the kinase activity and the intrinsic ligase activity are involved in the regulation of ubiquitination, strongly suggesting that Aurora-A mediates I{kappa}B{alpha} ubiquitination in vivo.


Figure 4105
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  		Figure 5. Aurora-A is an E3 ubiquitin ligase for I{kappa}B{alpha} in vitro and in vivo. (A) In vitro translated Aurora-A was incubated with I{kappa}B{alpha} in the presence or absence of UBE2N and I{kappa}B{alpha} ubiquitination was assessed with a total anti-I{kappa}B{alpha} antibody. (B) Recombinant Aurora-A, Aurora-A (K162M) and Aurora-B were incubated in the presence of K63 ubiquitin and UBE2N. GST- I{kappa}B{alpha} was added as a substrate. I{kappa}B{alpha} was detected by western blot using an anti-I{kappa}B{alpha} antibody. The membrane was re-probed with antibody against ubiquitin. The top panel shows the input of Aurora-A, Aurora-A (K162M) and Aurora-B. (C) Total I{kappa}B{alpha} was immunoprecipitated from MCF7 and MCF7 transfected with Aurora-A cells and probed with anti-ubiquitin antibody. The lower panels show the input of I{kappa}B{alpha} and Aurora-A.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
We have previously demonstrated that the E2 ubiquitin ligase UBE2N binds specifically to the Aurora-A Phe-31 variant in vitro and in vivo (11). In this study, we showed that UBE2N stimulates ubiquitination of Aurora-A both in vitro and in vivo. Further investigation of the Aurora-A/UBE2N association revealed a novel enzymatic function of Aurora-A resembling ubiquitin ligase. As in the case of MEKK1, kinase and ubiquitin ligase activity co-exist within the same protein. Similar findings have recently been reported for cyclin A/cdk2 (24), which facilitates p27 ubiquitination in a kinase-independent manner. It is not clear why a kinase would require ubiquitin ligase activity, but it is interesting that both MEKK1 and cyclin A/cdk2 mediate ubiquitination of their kinase substrates ERK2 and p27, respectively.

We presented evidence that the ubiquitin ligase activity of Aurora-A is dependent on the cysteines of the N-terminal domain. We examined the significance of three cysteine residues located at positions 8, 33 and 49, since most E3 ligases are characterized by the presence of critical cysteine residues distributed in particular sites on the protein. Mutational analysis of these residues showed their importance in contributing to Aurora-A ligase activity. Aurora-A does not have a canonical E3 domain such as the RING finger domain or a HECT domain, which might explain the ubiquitin ligase activity we observed. Since the 3D structure of full length Aurora-A is not yet available, the possibility that Aurora-A protein assumes a conformation that resembles a RING finger, which occurs in the case of U-box E3 ligases, cannot be assessed. Examination at the residues surrounding position 31 reveals the presence of a proline at position 32 (Fig. 3D), creating in the case of Aurora-A Phe-31 an FP motif. FP motifs have been classified as a newly identified CUE domain (19,25). The function of the CUE domain has been shown to be necessary for binding to either monoubiquitin or polyubiquitin chains. Although additional residues are also necessary for the CUE domain, the FP motif is essential and conserved. Moreover TAB2, a TAK1 interacting subunit also has a FP motif at its N-terminus, which is considered sufficient to classify it as a CUE domain containing protein (26). It is of interest that the two polymorphisms of Aurora-A, the Phe31Ile and Ile57Val are part of the CUE-like domain (Fig. 3B). Further examination of this variant is needed to elucidate its function at the molecular level. Recently, it has been shown that individuals with the Ile-31/Ile-57 variant of Aurora-A have an increased risk of oesophageal cancer (27). We also showed that the Aurora-A Phe-31 variant binds directly to and ubiquitinates I{kappa}B{alpha} in vitro and in vivo. I{kappa}B{alpha} colocalizes with Aurora-A at the centrosomes in mitotic cells. This dynamic subcellular localization of I{kappa}B{alpha} during the cell cycle and colocalization with Aurora-A in mitotic cells was observed in both cancer cells (HeLa, MCF7) and Rat1 fibroblasts (data not shown) to further support a general role for the interaction in cell growth control. In the classical NF-{kappa}B activation pathway, I{kappa}B{alpha} is phosphorylated on serine 32 and serine 36 by the I{kappa}B kinase (IKK) complex. The phosphorylated I{kappa}B{alpha} protein is subsequently ubiquitinated at residues lysine 21 and 22 via interaction with the SCFßTrCP ligase and degraded by the 26S proteosome (28–30). The ubiquitin chain type and the result of I{kappa}B{alpha} ubiquitination by Aurora-A is presently unknown, it may be required for its degradation or it may cause structural changes leading to NF-{kappa}B regulation. The significance of the association between Aurora-A and I{kappa}B{alpha} and its role in tumourigenesis needs to be further investigated.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Materials
Ubiquitin, methylated ubiquitin, MBP and Ubc10 were purchased from Sigma. Ubc5a, Ubc5b, Ubc13/Uev1a, Ubc3, Ubiquitin K48R, Ubiquitin K63 from Boston Biochemicals, GST-I{kappa}B{alpha} gift from Dr M. Karasarides, HA-UBE2N (C87A) gift from Dr ZJ Chen, GST- I{kappa}B{alpha} (amino acid 1-54) and GST- I{kappa}B{alpha} (amino acid 1-54) (Ser32A/Ser36A) gift from Dr DiDonato, HA-Ub gift from Dr P. Meier.

Cell culture and tranfection
HeLa and A549 cell lines were maintained in DMEM supplemented with 10% FCS, penicillin and streptomycin at 37°C and 5% CO2. For transient transfections, Lipofectamine Plus reagent (Invitrogen) was used according to manufacturer's instructions and cells were harvested 36 h post-transfection.

Protein production and purification and in vitro binding assay
For His-fusion proteins (Aurora-A, N-terminal Aurora-A (amino acid 1–117), C-terminal Aurora-A (amino acid 118–403), Aurora-A (K162M), UBE2N and MMS2 pRSET constructs were transformed into BL21(DE3)pLysS bacteria and streaked on ampicillin containing agar plates. A single colony was inoculated in 50 ml LB-ampicillin and incubated overnight at 37°C. After 24 h, 30 ml of the overnight culture were added to 500 ml of pre-warmed LB-ampicillin in a 2 l flask and the IPTG induction was followed at a final concentration of 1 mM. The flask was transferred into a 25°C incubator and growth was continued for 5 h. For the purification of His-tagged proteins, the pellet was lysed in ELB containing 1 mg/ml lysozyme and incubated on ice for 30 min. The lysed pellet was sonicated with six 10 s bursts at 200–300 W with a 10 s cooling period between each burst. DNase I (5 µg/ml) were added and incubated for 15 min on ice. The mix was then centrifuged at 10 000 g for 30 min at 4°C in order to pellet cellular debris and the supernatant was transferred to a fresh tube. 1 ml of Ni-NTA agarose was washed twice in lysis buffer and added to the supernatant. Following rotation at 4°C for 1 h, the lysate-Ni-NTA was loaded into a disposable column (Biorad) and after the beads settled they were rinsed twice in wash buffer (2x4 ml). The recombinant His-tagged protein was then collected by eluting four times with 0.5 ml elution buffer. The presence of the protein was detected with the Bradford reagent. The sequence coding for HA-Aurora-A and HA-Aurora-B proteins were cloned into BamH1 site of pFASTBac donor plasmid (GIBCO BRL, Life Technologies) using standard molecular biology techniques. The rest of the procedures were followed according to the protocol in the manufacturer's instruction (Bac-To-Bac® Baculovirus Expression Systems, GIBCO BRL, Life Technologies). Aurora-A or Aurora-B recombinant baculovirus was harvested after 72 h post-transfection incubation, tested for viral titre by plaque assay, and amplified by infecting a small volume (100–200 ml) of suspended Sf9 cell culture with multiplicity of infection (MOI) of 0.01–0.1. The amplified virus was harvested with 100-fold higher viral titre after 48–72 h post-infection incubation, and was used for protein production by infecting large quantity of suspended Sf9 cell culture (1 l) with MOI of 5–10 followed by 72–96 h post-infection incubation in the presence of okadaic acid (50 nM). Sf9 cells were then collected by centrifugation and the recombinant HA-Aurora-A and HA-Aurora-B proteins were purified from the cells by HA-affinity chromatography using protein G Sepharose 4FF (Pharmacia) beads coupled with {alpha}-HA antibody. The proteins were eluted using HA peptide (Roche Applied Science) according to manufacturer's instructions.

In vitro translation of proteins
1–3 µg of T7-containing cDNA plasmid was incubated with 40 µl of TNT QuickMaster Mix, 2 µl of cold or 35S methionine and the reaction was supplemented with H2O to make 50 µl. The mix was incubated at 30°C for 60–90 min.

Immunofluoresence
Cells were grown on 13 mM coverslips in six-well dishes. After appropriate inductions and/or transfections they were fixed in 4% paraformaldehyde in PBS for 30 min, permeabilized with 0.1% Triton-X in PBS for 10 min, rinsed and incubated with primary antibodies for an hour. After extensive washing, secondary antibody anti-mouse Alexia 568 or anti-rabbit Alexa 588 was added for 40 min. The cells were then washed and nuclei were stained with prior to mounting onto glass slides using Vectashield (Vector Laboratories). Optical sections were taken using a Leica TCS-SP2 confocal imaging system.

Immunoprecipitation and western blotting
Cells were lysed in lysis buffer (50 mM Hepes pH 7.5, 250 mM NaCl, 0.5% NP40 with protein inhibitors), incubated on ice for 30 min and centrifuged at 14 000 rpm for 15 min. The supernatant was collected and protein concentration was determined with Pierce BCA assay reagent. For western blotting, samples were separated on Novel Nupage Tris–Glycine gels (Invitrogen) followed by electrophoretic transfer on PVDF membrane (Millipore) and blocked with 5% non-fat milk. Incubation with primary and secondary antibodies were carried out at 4°C. Immunodetection was carried out using enhanced chemiluminescence detection (Amersham). For immunoprecipitation studies, lysates were incubated with the appropriate antibody in lysis buffer. After 2 h rotation at 4°C, Protein A or Protein G (Santa Cruz) was added for further overnight incubation. Samples were washed extensively with lysis buffer prior to resuspension in sample buffer. Antibodies used were IAK1 (Aurora-A) (Transduction Laboratories), ubiquitin and {gamma}-tubulin from Sigma, I{kappa}B{alpha} (C-21), GST, ezrin, HA, His and MYC purchased from Santa Cruz Biotechnology.

Generation of mutations using the quikchangeTM mutagenesis method (Aurora-A K162M and Aurora-A C8G, C33A, C47G)
QuikChangeTM mutagenesis was performed as described in the manufacturer's instructions. Briefly, complementary oligonucleotide primers containing the mutation were designed and used in a PCR reaction with Pfu polymerase and the wild-type plasmid DNA as a template. The reaction mixture was then digested with the restriction endonuclease Dpn I for 1 h at 37°C to remove the template DNA and transformed into competent JM109 bacteria. DNA prepared from the resulting colonies was sequenced to detect the presence and integrity of the mutation.

In vitro ubiquitination and kinase assay
For in vitro ubiquitination assays, the appropriate recombinant Aurora-A protein was incubated with E1 (Boston Biochemicals) at 50 nM, 5 µM recombinant UBE2N, UBE2N/MMS2 (1 : 1 ratio), 10 µM recombinant ubiquitin (Sigma) and 2 mM ATP in buffer containing an ATP regenerating system and 50 mM Tris pH 7.5, 0.05% NP40, 2.5 mM MgCl2. Reactions were carried out at 30°C for 1–57 h after which sample buffer was added before brief boiling and SDS-electrophoresis. In vitro kinase assays were performed in kinase buffer (50 mM Tris pH 7.5, 10 mM NaCl, 2.5 mM MgCl2, 1 mM DTT) containing {gamma}–P32-ATP and MBP substrate. The reactions were incubated for 30 min at 30°C and stopped by adding sample buffer. The reactions were separated on Novex Tris–Glycine gels and dried on a vacuum gel drier for 1 h at 80°C before exposure to Kodax-Biomax XR film.


   ACKNOWLEDGEMENTS
 
This work was funded by Program Grants from Breakthrough Breast Cancer and Cancer Research UK (CUK) (C309/A2187). We thank J. DiDonato, M. Karasarides, for the GST-I{kappa}B{alpha} constructs; Z.J. Chen for UBE2N (C87A) construct, P. Meier for the HA-Ub construct; A. Ashwort and P. Workman and all the members of the Cancer Drug Target Discovery Team for critically reading and discussing the manuscript.

Conflict of Interest statement. None declared.


   FOOTNOTES
 
{dagger} Present address: UCSF Comprehensive Cancer Center, PO Box 0875, 2340 Sutter St, San Francisco, CA 94143, USA. Back


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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