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Skp2 is required for Aurora B activation in cell mitosis and spindle checkpoint Juan Wu1,y,*, Yu-Fan Huang1,y, Xin-Ke Zhou2, Wei Zhang3, Yi-Fan Lian3, Xiao-Bin Lv4, Xiu-Rong Gao1, Hui-Kuan Lin5, Yi-Xin Zeng3, and Jian-Qing Huang1,* 1 Guangzhou Key Laboratory of Translational Medicine on Malignant Tumor Treatment; Affiliated Tumor Hospital of Guangzhou Medical University; Guangzhou, China; 2The Fifth Affiliated Hospital of Guangzhou Medical University; Guangzhou, China; 3State Key Laboratory of Oncology in South China and Department of Experimental Research; Sun Yat-Sen University Cancer Center; Guangzhou, China; 4Nanchang Key Laboratory of Cancer Pathogenesis and Translational Research; the Third Affiliated Hospital; Nanchang University; Nanchang, China; 5Department of Molecular and Cellular Oncology; The University of Texas M. D. Anderson Cancer Center; Houston, TX USA y
These authors contributed equally to this work.
Keywords: Aurora B, cell mitosis, genomic stability, Skp2, spindle checkpoint, tumorigenesis, ubiquitination Abbreviations: CENP-A, Centromere Protein-A; CPC, Chromosomal Passenger Complex; DDR, DNA Damage response; IF, Immunofluorescence; IB, Immunoblotting; IP, Immunoprecipitation; HR, Homologous Recombination; K, Lysine; MEFs, Mouse embryonic fibroblasts; SCF, Skp1-Cullin-F box protein; shRNA, small hairpin RNA; Skp2, S-phase kinase-associated protein 2; WB, Western Blot; WT, wild type.
The Aurora B kinase plays a critical role in cell mitosis and spindle checkpoint. Here, we showed that the ubiquitin E3ligase protein Skp2, also as a cell-cycle regulatory protein, was required for the activation of Aurora B and its downstream protein. When we restored Skp2 knockdown Hela cells with Skp2 and Skp2-LRR E3 ligase dead mutant we found that Skp2 could rescue the defect in the activation of Aurora B, but the mutant failed to do so. Furthermore, we discovered that Skp2 could interact with Aurora B and trigger Aurora B Lysine (K) 63-linked ubiquitination. Finally, we demonstrated the essential role of Skp2 in cell mitosis progression and spindle checkpoint, which was Aurora B dependent. Our results identified a novel ubiquitinated substrate of Skp2, and also indicated that Aurora B ubiquitination might serve as an important event for Aurora B activation in cell mitosis and spindle checkpoint.
Introduction Besides DNA repair machinery, successful mitosis is another barrier to maintain the integrity of genomic stability. During cell mitosis, the spindle assembly checkpoint ensures each daughter cell receives an exact copy of the whole genome. The Chromosomal Passenger Complex (CPC) comprising of protein including Aurora B, INCENP, Survivin, and Borealin, is an important regulator which coordinates exact chromosome segregation in the spindle assembly checkpoint. Among the CPC, Aurora B is the key kinase, inhibition of which leads to defect in spindle assembly checkpoint signaling.1,2 During early stage of mitosis, Aurora B localizes to centromeres and regulates kinetochore assembly, while in anaphase, Aurora B is ubiquitinated and dissociated from the centromeres, thus the cell can proceed to telophase and complete cytokinesis fluently.3 Skp2 belongs to the F box protein, which is a component of the Skp2 Skp1-Cullin-F box (SCF) E3 ligase complex. Skp2 serves as a cell-cycle regulator by targeting cell-cycle inhibitors such as p27 and p21 for ubiquitination and subsequent degradation.4-6 As well as the critical role in cell-cycle progression, Skp2
also displays oncogenic activity.4,5,7 Furthermore, our recent study revealed that Skp2 also played an important role in DNA Damage response (DDR) by regulating ATM kinase and Homologous Recombination (HR) repair.8 As the 2 main monitoring machineries, lots of evidence indicates that there exists crosstalk between mitosis regulation and DNA repair machinery.9-11 A recent study reported that as the core kinase of the DDR ATM was activated in mitosis in the absence of DNA damage, which was dependent on the Aurora-B kinase activity.9 Therefore, it makes us wonder whether Skp2, another ATM regulator in DDR, is also involved in the cell mitosis progression. In this study, we aim to examine the role of Skp2 in cell mitosis and spindle checkpoint. We show that Skp2 is required for activation of Aurora B and its downstream protein. We also discover that Skp2 can interact with Aurora B and trigger Aurora B ubiquitination. Furthermore, just like Aurora B, Skp2 is also verified to play an essential role in cell mitosis and spindle checkpoint. We propose that Skp2 regulates Aurora B activation by ubiquitination in cell mitosis and spindle checkpoint.
*Correspondence to: Juan Wu; Email: [email protected]; Jian-Qing Huang; Email: [email protected] Submitted: 05/20/2015; Revised: 10/11/2015; Accepted: 11/12/2015 http://dx.doi.org/10.1080/15384101.2015.1120916
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Results
Figure 1. Skp2 is required for activation of Aurora B in cell mitosis. (A) HeLa luciferase knockdown control cells transfected with p27 or not, and Skp2 knockdown cells were synchronized by nocodazole for 18 hrs before harvested. Mitotic HeLa cells were collected by mitotic shakeoff. The collected cells were then continued to incubate for the indicated time points (0, 1, 3 hrs) and following immunoblotted with indicated antibodies. Since the ectopic expression of p27 was too strong to see the endogenous p27 clearly, we rerun the last 9 lanes and incubated with p27 and GAPDH antibody again. (C) HeLa cells with luciferase control or Skp2 knockdown were synchronized by nocodazole for 18 hrs before harvested. Mitotic HeLa cells were collected by mitotic shakeoff. The collected cells were then continued to incubate for the indicated time points (0, 1, 2, 3 hrs) and following immunoblotted with indicated antibodies. (B, D) HeLa cells with luciferase control or Skp2 knockdown were synchronized by nocodazole for 18 hrs and then were fixed for IF analysis with indicated antibodies. (E) HeLa cells with luciferase control (mock), Skp2 knockdown restored with vector control, Xp-Skp2 or Skp2-LRR plasmids were synchronized by nocodazole for 18 hrs. Mitotic HeLa cells were collected by mitotic shakeoff. The collected cells were then continued to incubate for the indicated time points (0, 1, 3 hrs), followed by western blot analysis with indicated antibodies.
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Skp2 is required for activation of Aurora B in cell mitosis Besides the role in cell-cycle checkpoint, tumorigenesis and cancer metastasis, our previous study showed that Skp2 could regulate ATM activation and subsequently play a pivotal role in DDR,8 indicating that Skp2 is also required for the maintenance of genomic stability. As cell mitosis is another machinery to maintain genomic stability, we speculate Skp2 may be also related to cell mitosis. Aurora-B is the key kinase in cell mitosis, and importantly, Aurora-B also regulates ATM activation in the process of cell mitosis, which is independent on DDR.9 To this end, we first wonder whether Skp2 affect Aurora B kinase activity. As is the activated form of Aurora B, we examined the phospho-Aurora B level by Western Blot (WB) in Skp2 and control knockdown Hela cells, which were synchronized by nocodazole or released to cell cycle again by further incubating without nocodazole for different time periods. We found that phospho-Aurora B was impaired in Skp2 knockdown cells compared to the control no whether cells were synchronized or released to cell cycle at different time points (Fig. 1A). Consistently, using immunofluorescence (IF) assay, we discovered the same impairment of phospho-Aurora B in Skp2 knockdown synchronized cells (Fig. 1B). As Skp2 regulates G2/M phase progression by mediating the degradation of p27,12 in the WB assay we also included p27 as a positive control. As expected p27 increased upon Skp2 knockdown, and opposite to phosphoAurora B, with cell cycle release p27 level further went up. Furthermore, when we overexpressed p27, phospho-Aurora B decreased (Fig. 1A). Since phosphorylation of centromere protein-A (CENP-A) is regulated by Aurora B kinase,13 we also measured its level in the same condition. No matter through WB test or IF assay, the phospho-CENP-A level decreased in the Skp2 knockdown cells compared to the control (Fig. 1C and 1D). To determine the role of Skp2 E3 ligase activity in the activation of Aurora
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B in cell mitosis, we applied the Skp2-LRR E3 ligase dead mutant devoid of the N-terminus and F-box domain as well as Skp2 wild type (WT) to restore Skp2 knockdown Hela cells and analyzed Aurora B activation (Fig. 1E). The results showed that while Skp2 WT could rescue the activity of Aurora B and its substrate CENP-A, but the Skp2-LRR mutant failed to do so, indicating that the E3 ligase activity of Skp2 is essential for its role in activating Aurora B. Our results suggest that Skp2 is required for the activation of Aurora B in cell mitosis.
level of which dramatically decreased in Skp2 knockdown cells (Fig. 3C). Since Cul1 is another important component like Skp2 to constitute the Skp2 SCF complex, we knocked down Cul1 in 293T cells and performed ubiquitination assay again. As expected Cul1 knockdown could inhibit Skp2-mediated Aurora B ubiquitination (Fig. 3D). Skp2 is known to target protein for ubiquitination and subsequent degradation.4-6 However, Skp2 induced Aurora B ubiquitination, but it didn’t cause Aurora B
Skp2 and Aurora B interact with each other Having shown that Skp2 is involved in the activation of Aurora B in cell mitosis, we first speculate that Skp2 and Aurora B may interact with each other. In order to explore this, we performed a series of coimmunoprecipitation (Co-IP) assay. As predicted, we found that Skp2 and Aurora B interacted with each other exdogenously by reciprocal immunoprecipitation (IP) (Fig. 2A and 2B). Furthermore, Skp2 was also shown to interact with Aurora B endogenously (Fig. 2C). IF of mitotic cells showed that phospho-Aurora B and Skp2 localize similarly mainly at the cellplasm in early mitosis (Fig. 2D). These data further demonstrated the intimate relationship between Skp2 and Aurora B. Skp2 triggers Aurora B ubiquitination As shown that Skp2 interacts with Aurora B and is required for its activation in the process of cell mitosis, we next seek to determine the mechanism how Skp2 regulates Aurora B activation. As aforementioned, Skp2 is known to target protein for ubiquitination, and Aurora B can be ubiquitinated by Cul3/KLHL9/KLHL13 ligase,3 we next want to know whether Skp2 also regulates Aurora B ubiquitination. Indeed, we found that Skp2 remarkably triggered in vivo ubiquitination of Aurora B (Fig. 3A). In further support of this notion, we also did the ubiquitination assay in Skp2 knockdown cells. Compared to the control, the basal level of Aurora B ubiquitination decreased in Skp2 knockdown cells (Fig. 3B). Moreover, we also detected endogenous Aurora B ubiquitination in Hela cells, while the
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Figure 2. Skp2 and Aurora B interact with each other. (A) 293T cells were transfected with XpSkp2 and/or HA-Aurora B, and cells were harvested for coimmmunoprecipitation assay with HA antibody, followed by western blot analysis. (B) 293T cells were transfected with Flag-Skp2 and/or HAAurora B, and cells were harvested for coimmmunoprecipitation assay with Flag antibody, followed by western blot analysis. (C) 293T cells with luciferase control or Skp2 knockdown were harvested and coimmmunoprecipitated with Skp2 antibody, followed by western blot analysis. (D) HeLa cells were transfected with Xp-Skp2, and then were synchronized by nocodazole for 18 hrs, followed by IF analysis with Xpress antibody and phospho-Aurora B antibody.
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Skp2 is essential for cell mitosis progression and spindle checkpoint An earlier study demonstrated that hepatocytes from Skp2¡/¡ mice displayed polyploidy compared to WT mice.14 Here we further confirmed this phenomenon in Skp2¡/¡ MEF cells (Fig. 4A). Since Aurora B is a key kinase in cell mitosis and spindle checkpoint, and our data show that Skp2 is essential for aurora B kinase activity, we next aim to explore the role of Skp2 in cell mitosis and spindle checkpoint. In order to achieve this goal, we first generated stable HeLa cellline with luciferase control and Skp2 knockdown clone #1 and #2 (Fig. 4B). Compared to luciferase control, more cells were arrested in G2/M phase upon Skp2 knockdown after nacodazole treatment (Fig. 4C), also consistent with knockdown efficiency, implying that Skp2 deficiency leads to defect in cell mitosis. In order to further monitor the cell mitotic dynamics, we analyzed the same set of luciferase control and Skp2 knockdown Hela cells with a time-lapse microscopy. We found that it took more time for Skp2 knockdown cells to progress into anaphase compared with control, especially for Skp2 shRNA#1, whose knockdown efficiency was better. For the control cells, the time between nuclear Figure 3. Skp2 triggers Aurora B ubiquitination. (A) 293T cells were transfected with the indicated plasmids envelope breakdown and anaphase and harvested for in vivo ubiquitination assay (see the Materials and Methods for details). (B) 293T cells onset was around 48 mins, while with luciferase control or Skp2 knockdown were transfected with the indicated plasmids and harvested for the time for Skp2 shRNA#1 and in vivo ubiquitination assay. (C) 293T cells with luciferase control or Skp2 knockdown were harvested and shRNA#2 was around 59 and coimmmunoprecipitated with Aurora B antibody, followed by western blot analysis with ubiquitin antibody. (D) 293T cells with Cul-1 or luciferase control knockdown were transfected with indicated plasmids 56 mins independently (Fig. 4E). and harvested for in vivo ubiquitination assay. (E) 293T cells were transfected with HA-Aurora B, Xp-Skp2, As Skp2 interacts with Aurora B along with His-Ub WT, K48R, or K63R mutant and harvested for in vivo ubiquitination assay. and triggers its ubiquitination, we expect that Skp2-mediated cell mitosis progression is Aurora B degradation (Fig. 3A). It indicates that Skp2-induced Aurora B dependent. Therefore we also overexpress Aurora B in Skp2 ubiquitination may be through Lysine (K) 63-linked. Indeed we knockdown clone#1 cells in the Flow Cytometry assay and the found that K63 mutation could impair Aurora B ubiquitination Live-Cell Time-Lapse Imaging experiment. As anticipated, with but Lysine (K) 48 mutation could not (Fig. 3E). Therefore, our Aurora B overexpression, less mitotic cells were arrested in G2/M results suggest that Skp2-mediated Aurora B ubiquitination phase (14.6% vs 28.2%), and the metaphase time came back to may play an important role for the interaction of Skp2 and around 49 mins (Fig. 4D and Fig. 4E), indicating that ectopic Aurora B and the subsequent activation of Aurora B in cell expression of Aurora B in Skp2-depleted cells could partially restore the mitotic defects. Taken together, these data strongly mitosis and spindle checkpoint.
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Figure 4. For figure legend, see page 3882.
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demonstrate the essential role of Skp2 in cell mitosis progression and spindle checkpoint, which is Aurora B dependent.
Discussion CPC is required for correct spindle-kinetochore attachments during chromosome segregation and cytokinesis, of which Aurora B is the key kinase. Deficiency of Aurora B makes the mitotic cells stay at anaphase and can’t proceed to form an organized anaphase spindle and subsequently complete cytokinesis.3 Thus, Aurora B plays a central role in cell mitosis progression and spindle checkpoint. Skp2 is a multifunctional protein. It’s known to play a critical role in cell cycle regulation,6,12 and also demonstrated to contribute to tumorigenesis, cancer development and metastasis in recent years.5,7,15,16 Since Skp2 deficiency results in polyploidy and centrosome overduplication,14 it suggests that Skp2 may be required to maintain genomic stability. Prominently, we have recently demonstrated that Skp2 induced NBS ubiquitination, in turn regulating activation and recruitment of ATM to DNA damage foci in DDR. Furthermore, Skp2 knockdown was shown to exhibit defect in HR repair.8 This important finding further verifies the pivotal role of Skp2 in DDR and maintaining genomic stability. In this study, we provide novel insight into how Skp2 take part in maintaining genomic stability in the process of cell mitosis. We show that Skp2 is required for the activation of Aurora B and its downstream protein, which is the key kinase in cell mitosis and spindle checkpoint. Moreover, Skp2 and Aurora B are shown to interact with each other. Furthermore, Skp2 is also demonstrated to play an essential role in cell mitosis progression and spindle checkpoint, which is Aurora B dependent. Importantly, the E3 ligase activity of Skp2 is required for its role in activating Aurora B. We further demonstrate that Skp2 triggers K63linked ubiquitination of Aurora B. Ubiquitination has multiple important functions including cell cycle control, apoptosis, immune functions, as well as neuron degeneration.17-19 Traditionally, ubiquitination is always targeting protein for degradation. Very recently, protein ubiquitination is also shown to play non-proteolytic functions such as protein trafficking, DNA damage repair as well as protein kinase activation.8,17,20-22 Besides the
role in cell cycle regulation by targeting protein for degradation like p21 and p27, Skp2 is also demonstrated to play central role in DDR and tumorigenesis through kinase activation followed by K63-linked ubiquitination of NBS1 and Akt.7,8 Therefore, in this study Skp2 mediated K63-linked ubiquitination of Aurora B may be responsible for Aurora B kinase activation in cell mitosis and spindle checkpoint. Given that K63-linked ubiquitination is recently shown to provide a docking site for protein-protein interaction,8,17,23 it is very likely that Aurora B ubiquitination may enable the interaction of Skp2 and Aurora B, in turn facilitating activation of Aurora B and its downstream protein. In order to better understand the role of Aurora B ubiquitination, future study will be important to identify the Aurora B ubiquitination sites for Skp2. ATM occupies the central node of the DDR network, thus plays an important role in controlling genomic stability. ATM functions by phosphorylating a list of downstream targets to facilitate cell-cycle checkpoint in response to DDR.24 Interestingly, in the progression of cell mitosis ATM phosphorylates a different but overlapping list of substrates with DDR. For example, some mitotic spindle checkpoint proteins, such as Bub1, Mad1, and Sgo1 are also among the substrates in DDR.25 All these evidence demonstrates that there exists crosstalk between mitotic regulatory machinery and DDR pathway. As is shown recently that Aurora B phosphorylates ATM, and it’s essential for ATM activation in the process of cell mitosis.9 Furthermore, our finding also demonstrates that ATM is a downstream target of Skp2 in the DDR.8 Meanwhile, in this study we found that Aurora B is a novel substrate for Skp2 ubiquitination in cell mitosis. Our study further supports the notion that mitotic checkpoint shares some common molecular components with the DDR pathway. Thus, in order to maintain genomic stability, Skp2 is also involved in the mitotic checkpoint dependent on Aurora B. Given that Skp2 overexpression is a poor prognosis factor for cancer patients and also shown to be critical for the sensitivity of cancer cells to radiotherapy in some cancer cell lines,8,26 Skp2 is recognized as a promising target for cancer treatment. Certain chemical target of Skp2 is also validated to inhibit tumor progression in lung and prostate cancer models.27 Here we show that Skp2 regulates Aurora B activation in the mitosis progression, probably through its ubiquitinating function, and Skp2
Figure 4. (See previous page) Skp2 is essential for cell mitosis progression and spindle checkpoint. (A) Wide Type (WT) and Skp2¡/¡ MEFs were harvested for cell cycle analysis. The left panel shows the representative flow cytometry profile and the right panel displays the statistical results. Error bars represent the standard deviation. (B) Skp2 level was shown for the luciferase control and Skp2 knockdown clone#1 and #2 HeLa cells, which were used for the following FASCS assay (C, D) and Live cell time-lapse imaging assay (E). (C) HeLa cells with luciferase control or Skp2 knockdown were synchronized by nocodazole for 18 hrs and then all the cells were harvested for FASCS assay. The mitotic index was measured based on Histone H3S10p staining. The left panel shows the representative flow cytometry profile and the right panel displays the statistical results. Error bars represent the standard deviation. (D) Skp2 knockdown clone#1 HeLa cells were transfected with vector control or HA-Aurora B plasmids for 30 hrs, synchronized by nocodazole for 18 hrs and then all the cells were harvested for FASCS assay. The mitotic index was measured based on Histone H3S10p staining. The left panel shows the representative flow cytometry profile, the middle panel displays the statistical results, and the right panel shows the Aurora B expression level of the cells used in the assay. Error bars represent the standard deviation. (E) Luciferase control or Skp2 knocking-down HeLa cells expressing H2B-GFP were imaged at every 5 minutes, and Skp2 knockdown clone#1 HeLa cells expressing H2B-GFP transfected with pmCherry-C1-Aurora B was also included. The left panel shows the representative fluorescence video microscopy series from the onset of mitosis to monitor chromosome dynamics and the right panel displays the statistical results. Cell numbers were 100 (Control shRNA), 94(Skp2 shRNA#1), 100(Skp2 shRNA#2), and 72(Skp2 shRNA#1+ Aurora B). Error bars represent the standard deviation.
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deficiency inhibits cell mitosis progression and cytokinesis, which further identifies the function of Skp2 in maintaining genomic stability and subsequently the role in tumorigenesis. Therefore, our data provides new evidence to identify Skp2 as a target for cancer treatment.
were cultured in the medium containing 2mg/ml puromycin for 1 week. The following lentiviral shRNAs were used for transfection: Skp2-lentiviral shRNA#1 (50 -GATAGTGTCATGCTAAAGAAT-3’), Skp2-lentiviral shRNA#2 (50 -GCCTAAGCTAAATCGAGAGAA-3’), Cul1-lentiviral shRNA (50 CCCGCAGCAAATA GTTCATGT-3’).
Materials and Methods
In vivo ubiquitination assay In vivo ubiquitination assay was performed as described.20 In brief, 293T cells were transfected with the indicated plasmids for 48 hrs and harvested by denatured buffer (6 M guanidine-HCl, 0.1 M Na2HPO4/NaH2PO4, 10mMimidazole). The cell extracts were then incubated with nickel beads (Ni-NTA) for 3 hrs, washed, and subjected to western blot analysis.
Cell culture and reagents Mouse embryonic fibroblasts (MEFs) from wild-type and Skp2¡/¡ mice were prepared as previously described.16 293T, HeLa cells were cultured in DMEM containing 10% fetal bovine serum (FBS). To clone Xp-Skp2, Skp2 was amplified by PCR using pcDNA3-Skp2 as a template and inserted into a pcDNA4/ HisMax-TOPO vector (Invitrogen). (His)6-ubiquitin, (His)6ubiquitin K48R, and (His)6-ubiquitin K63R were previously described.20 HA-Aurora B was a gift from Dr. Yi Liang. To clone pmCherry-C1-Aurora B plasmid, Aurora B was amplified by PCR with HA-Aurora B as a template and inserted into the pmCherry-C1 vector. Immunoprecipitation (IP), immunoblotting (IB), and immunofluorescence(IF) IP, IB, and IF were done essentially as described with mild modification.5,8 For IP, cells were lysed by E1A lysis buffer [250 mM NaCl, 50 mM HEPES (pH 7.5), 0.1% NP-40, 5 mM EDTA, protease inhibitor cocktail (Roche)]. The following antibodies were used for IP, IB, and IF: Skp2 antibody (IP: 1:200, IB: 1:2000, Invitrogen), Xpress antibody (IP: 1:500; IB: 1:5000, Invitrogen), Flag antibody (M2) (IP: 1:200; IB: 1:3000, Sigma), Cul-1 antibody (IB: 1:1000, Invitrogen), HA antibody (IP: 1:200; IB: 1:5000, Cell Signal), Aurora B phospho T232 (IF: 1:200; IB: 1:1000, Abcam), phospho-CENP-A (IF: 1:200; IB: 1:1000, Cell Signal). Viral infection For lentiviral short hairpin RNA (shRNA) infection, 293T cells were co-transfected with shRNAs against luciferase or Skp2 along with packing plasmids (deltaVPR8.9) and envelope plasmid (VSVG) using the calcium phosphate precipitation method. Two days after transfection, virus particles containing Skp2 and luciferase shRNAs were used to infect mammalian cells. All the infected cells References 1. Ditchfield C, Johnson VL, Tighe A, Ellston R, Haworth C, Johnson T, Mortlock A, Keen N, Taylor SS. Aurora B couples chromosome alignment with anaphase by targeting BubR1, Mad2, and Cenp-E to kinetochores. J Cell Biol 2003; 161:267-80; PMID:12719470; http://dx.doi.org/10.1083/ jcb.200208091 2. Hauf S, Cole RW, LaTerra S, Zimmer C, Schnapp G, Walter R, Heckel A, van Meel J, Rieder CL, Peters JM. The small molecule Hesperadin reveals a role for Aurora B in correcting kinetochore-microtubule attachment and in maintaining the spindle assembly checkpoint. J Cell Biol 2003; 161:281-94; PMID:12707311; http://dx.doi.org/10.1083/ jcb.200208092
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Flow cytometry For mitotic arrest, Hela cells were synchronized by 200 ng/ml nocodazole for 18 hrs. For flow cytometry analysis, cells were then collected and permeabilized with 0.1% Triton X-100 after fixation in 75% ethanol, stained with Histone H3p antibody (Millipore) and FITC-conjugated second antibody. For DNA content, cells were stained with propidium iodide (PI) (Sigma). After that analysis were performed with a FACS flow cytometer. For Cell-Cycle Analysis, WT and Skp2¡/¡ MEFs cultured in 10% FBS were fixed in 75% ethanol, and stained with PI, followed by flow cytometry analysis. Live-cell time-lapse imaging To monitor chromosome dynamics, GFP-tagged H2B was transfected into HeLa cells with luciferase control or Skp2 knockdown transfected with Aurora B or not. 24 hrs later, cells were imaged at 5min intervals for 24h using live-cell imaging. Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed. Funding
This work was supported by the National Natural Science Foundation of China (81201550), Zhuangjiang Star 2015 of Science and Technology Program of Guangzhou, China (201506010037) to Juan Wu.
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and p27(Kip1), polyploidy and centrosome overduplication. EMBO J 2000; 19:2069-81; PMID:10790373; http://dx.doi.org/10.1093/emboj/19.9.2069 Chan CH, Lee SW, Li CF, Wang J, Yang WL, Wu CY, Wu J, Nakayama KI, Kang HY, Huang HY, et al. Deciphering the transcriptional complex critical for RhoA gene expression and cancer metastasis. Nat Cell Biol 2010; 12:457-67; PMID:20383141; http://dx.doi. org/10.1038/ncb2047 Lin HK, Chen Z, Wang G, Nardella C, Lee SW, Chan CH, Yang WL, Wang J, Egia A, Nakayama KI, et al. Skp2 targeting suppresses tumorigenesis by Arf-p53independent cellular senescence. Nature 2010; 464:374-9; PMID:20237562; http://dx.doi.org/ 10.1038/nature08815 Bhoj VG, Chen ZJ. Ubiquitylation in innate and adaptive immunity. Nature 2009; 458:430-7; PMID:19325622; http://dx.doi.org/10.1038/ nature07959 Giasson BI, Lee VM. Are ubiquitination pathways central to Parkinson disease? Cell 2003; 114:1-8; PMID:12859888; http://dx.doi.org/10.1016/S00928674(03)00509-9 Hoeller D, Dikic I. Targeting the ubiquitin system in cancer therapy. Nature 2009; 458:438-44; PMID:19325623; http://dx.doi.org/10.1038/ nature07960 Yang WL, Wang J, Chan CH, Lee SW, Campos AD, Lamothe B, Hur L, Grabiner BC, Lin X, Darnay BG, et al. The E3 ligase TRAF6 regulates Akt ubiquitination and activation. Science 2009; 325:1134-8; PMID:19713527; http://dx.doi.org/10.1126/ science.1175065
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21. Chen ZJ, Sun LJ. Nonproteolytic functions of ubiquitin in cell signaling. Mol Cell 2009; 33:275-86; PMID:19217402; http://dx.doi.org/10.1016/j.molcel. 2009.01.014 22. Raiborg C, Stenmark H. The ESCRT machinery in endosomal sorting of ubiquitylated membrane proteins. Nature 2009; 458:445-52; PMID:19325624; http:// dx.doi.org/10.1038/nature07961 23. Yang WL, Wu CY, Wu J, Lin HK. Regulation of Akt signaling activation by ubiquitination. Cell Cycle 2010; 9:487-97; PMID:20081374 24. Kastan MB, Bartek J. Cell-cycle checkpoints and cancer. Nature 2004; 432:316-23; PMID:15549093; http://dx.doi.org/10.1038/nature03097 25. Matsuoka S, Ballif BA, Smogorzewska A, McDonald ER, 3rd, Hurov KE, Luo J, Bakalarski CE, Zhao Z, Solimini N, Lerenthal Y, et al. ATM and ATR substrate analysis reveals extensive protein networks responsive to DNA damage. Science 2007; 316:11606; PMID:17525332; http://dx.doi.org/10.1126/ science.1140321 26. Harada K, Supriatno, Kawaguchi S, Kawashima Y, Itashiki Y, Yoshida H, Sato M. High expression of S-phase kinase-associated protein 2 (Skp2) is a strong prognostic marker in oral squamous cell carcinoma patients treated by UFT in combination with radiation. Anticancer Res 2005; 25:2471-5; PMID:16080478 27. Chan CH, Morrow JK, Li CF, Gao Y, Jin G, Moten A, Stagg LJ, Ladbury JE, Cai Z, Xu D, et al. Pharmacological inactivation of Skp2 SCF ubiquitin ligase restricts cancer stem cell traits and cancer progression. Cell 2013; 154:556-68; PMID:23911321; http://dx. doi.org/10.1016/j.cell.2013.06.048
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Skp2 is required for Aurora B activation in cell mitosis and spindle checkpoint Juan Wu1,y,*, Yu-Fan Huang1,y, Xin-Ke Zhou2, Wei Zhang3, Yi-Fan Lian3, Xiao-Bin Lv4, Xiu-Rong Gao1, Hui-Kuan Lin5, Yi-Xin Zeng3, and Jian-Qing Huang1,* 1 Guangzhou Key Laboratory of Translational Medicine on Malignant Tumor Treatment; Affiliated Tumor Hospital of Guangzhou Medical University; Guangzhou, China; 2The Fifth Affiliated Hospital of Guangzhou Medical University; Guangzhou, China; 3State Key Laboratory of Oncology in South China and Department of Experimental Research; Sun Yat-Sen University Cancer Center; Guangzhou, China; 4Nanchang Key Laboratory of Cancer Pathogenesis and Translational Research; the Third Affiliated Hospital; Nanchang University; Nanchang, China; 5Department of Molecular and Cellular Oncology; The University of Texas M. D. Anderson Cancer Center; Houston, TX USA y
These authors contributed equally to this work.
Keywords: Aurora B, cell mitosis, genomic stability, Skp2, spindle checkpoint, tumorigenesis, ubiquitination Abbreviations: CENP-A, Centromere Protein-A; CPC, Chromosomal Passenger Complex; DDR, DNA Damage response; IF, Immunofluorescence; IB, Immunoblotting; IP, Immunoprecipitation; HR, Homologous Recombination; K, Lysine; MEFs, Mouse embryonic fibroblasts; SCF, Skp1-Cullin-F box protein; shRNA, small hairpin RNA; Skp2, S-phase kinase-associated protein 2; WB, Western Blot; WT, wild type.
The Aurora B kinase plays a critical role in cell mitosis and spindle checkpoint. Here, we showed that the ubiquitin E3ligase protein Skp2, also as a cell-cycle regulatory protein, was required for the activation of Aurora B and its downstream protein. When we restored Skp2 knockdown Hela cells with Skp2 and Skp2-LRR E3 ligase dead mutant we found that Skp2 could rescue the defect in the activation of Aurora B, but the mutant failed to do so. Furthermore, we discovered that Skp2 could interact with Aurora B and trigger Aurora B Lysine (K) 63-linked ubiquitination. Finally, we demonstrated the essential role of Skp2 in cell mitosis progression and spindle checkpoint, which was Aurora B dependent. Our results identified a novel ubiquitinated substrate of Skp2, and also indicated that Aurora B ubiquitination might serve as an important event for Aurora B activation in cell mitosis and spindle checkpoint.
Introduction Besides DNA repair machinery, successful mitosis is another barrier to maintain the integrity of genomic stability. During cell mitosis, the spindle assembly checkpoint ensures each daughter cell receives an exact copy of the whole genome. The Chromosomal Passenger Complex (CPC) comprising of protein including Aurora B, INCENP, Survivin, and Borealin, is an important regulator which coordinates exact chromosome segregation in the spindle assembly checkpoint. Among the CPC, Aurora B is the key kinase, inhibition of which leads to defect in spindle assembly checkpoint signaling.1,2 During early stage of mitosis, Aurora B localizes to centromeres and regulates kinetochore assembly, while in anaphase, Aurora B is ubiquitinated and dissociated from the centromeres, thus the cell can proceed to telophase and complete cytokinesis fluently.3 Skp2 belongs to the F box protein, which is a component of the Skp2 Skp1-Cullin-F box (SCF) E3 ligase complex. Skp2 serves as a cell-cycle regulator by targeting cell-cycle inhibitors such as p27 and p21 for ubiquitination and subsequent degradation.4-6 As well as the critical role in cell-cycle progression, Skp2
also displays oncogenic activity.4,5,7 Furthermore, our recent study revealed that Skp2 also played an important role in DNA Damage response (DDR) by regulating ATM kinase and Homologous Recombination (HR) repair.8 As the 2 main monitoring machineries, lots of evidence indicates that there exists crosstalk between mitosis regulation and DNA repair machinery.9-11 A recent study reported that as the core kinase of the DDR ATM was activated in mitosis in the absence of DNA damage, which was dependent on the Aurora-B kinase activity.9 Therefore, it makes us wonder whether Skp2, another ATM regulator in DDR, is also involved in the cell mitosis progression. In this study, we aim to examine the role of Skp2 in cell mitosis and spindle checkpoint. We show that Skp2 is required for activation of Aurora B and its downstream protein. We also discover that Skp2 can interact with Aurora B and trigger Aurora B ubiquitination. Furthermore, just like Aurora B, Skp2 is also verified to play an essential role in cell mitosis and spindle checkpoint. We propose that Skp2 regulates Aurora B activation by ubiquitination in cell mitosis and spindle checkpoint.
*Correspondence to: Juan Wu; Email: [email protected]; Jian-Qing Huang; Email: [email protected] Submitted: 05/20/2015; Revised: 10/11/2015; Accepted: 11/12/2015 http://dx.doi.org/10.1080/15384101.2015.1120916
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Results
Figure 1. Skp2 is required for activation of Aurora B in cell mitosis. (A) HeLa luciferase knockdown control cells transfected with p27 or not, and Skp2 knockdown cells were synchronized by nocodazole for 18 hrs before harvested. Mitotic HeLa cells were collected by mitotic shakeoff. The collected cells were then continued to incubate for the indicated time points (0, 1, 3 hrs) and following immunoblotted with indicated antibodies. Since the ectopic expression of p27 was too strong to see the endogenous p27 clearly, we rerun the last 9 lanes and incubated with p27 and GAPDH antibody again. (C) HeLa cells with luciferase control or Skp2 knockdown were synchronized by nocodazole for 18 hrs before harvested. Mitotic HeLa cells were collected by mitotic shakeoff. The collected cells were then continued to incubate for the indicated time points (0, 1, 2, 3 hrs) and following immunoblotted with indicated antibodies. (B, D) HeLa cells with luciferase control or Skp2 knockdown were synchronized by nocodazole for 18 hrs and then were fixed for IF analysis with indicated antibodies. (E) HeLa cells with luciferase control (mock), Skp2 knockdown restored with vector control, Xp-Skp2 or Skp2-LRR plasmids were synchronized by nocodazole for 18 hrs. Mitotic HeLa cells were collected by mitotic shakeoff. The collected cells were then continued to incubate for the indicated time points (0, 1, 3 hrs), followed by western blot analysis with indicated antibodies.
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Skp2 is required for activation of Aurora B in cell mitosis Besides the role in cell-cycle checkpoint, tumorigenesis and cancer metastasis, our previous study showed that Skp2 could regulate ATM activation and subsequently play a pivotal role in DDR,8 indicating that Skp2 is also required for the maintenance of genomic stability. As cell mitosis is another machinery to maintain genomic stability, we speculate Skp2 may be also related to cell mitosis. Aurora-B is the key kinase in cell mitosis, and importantly, Aurora-B also regulates ATM activation in the process of cell mitosis, which is independent on DDR.9 To this end, we first wonder whether Skp2 affect Aurora B kinase activity. As is the activated form of Aurora B, we examined the phospho-Aurora B level by Western Blot (WB) in Skp2 and control knockdown Hela cells, which were synchronized by nocodazole or released to cell cycle again by further incubating without nocodazole for different time periods. We found that phospho-Aurora B was impaired in Skp2 knockdown cells compared to the control no whether cells were synchronized or released to cell cycle at different time points (Fig. 1A). Consistently, using immunofluorescence (IF) assay, we discovered the same impairment of phospho-Aurora B in Skp2 knockdown synchronized cells (Fig. 1B). As Skp2 regulates G2/M phase progression by mediating the degradation of p27,12 in the WB assay we also included p27 as a positive control. As expected p27 increased upon Skp2 knockdown, and opposite to phosphoAurora B, with cell cycle release p27 level further went up. Furthermore, when we overexpressed p27, phospho-Aurora B decreased (Fig. 1A). Since phosphorylation of centromere protein-A (CENP-A) is regulated by Aurora B kinase,13 we also measured its level in the same condition. No matter through WB test or IF assay, the phospho-CENP-A level decreased in the Skp2 knockdown cells compared to the control (Fig. 1C and 1D). To determine the role of Skp2 E3 ligase activity in the activation of Aurora
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B in cell mitosis, we applied the Skp2-LRR E3 ligase dead mutant devoid of the N-terminus and F-box domain as well as Skp2 wild type (WT) to restore Skp2 knockdown Hela cells and analyzed Aurora B activation (Fig. 1E). The results showed that while Skp2 WT could rescue the activity of Aurora B and its substrate CENP-A, but the Skp2-LRR mutant failed to do so, indicating that the E3 ligase activity of Skp2 is essential for its role in activating Aurora B. Our results suggest that Skp2 is required for the activation of Aurora B in cell mitosis.
level of which dramatically decreased in Skp2 knockdown cells (Fig. 3C). Since Cul1 is another important component like Skp2 to constitute the Skp2 SCF complex, we knocked down Cul1 in 293T cells and performed ubiquitination assay again. As expected Cul1 knockdown could inhibit Skp2-mediated Aurora B ubiquitination (Fig. 3D). Skp2 is known to target protein for ubiquitination and subsequent degradation.4-6 However, Skp2 induced Aurora B ubiquitination, but it didn’t cause Aurora B
Skp2 and Aurora B interact with each other Having shown that Skp2 is involved in the activation of Aurora B in cell mitosis, we first speculate that Skp2 and Aurora B may interact with each other. In order to explore this, we performed a series of coimmunoprecipitation (Co-IP) assay. As predicted, we found that Skp2 and Aurora B interacted with each other exdogenously by reciprocal immunoprecipitation (IP) (Fig. 2A and 2B). Furthermore, Skp2 was also shown to interact with Aurora B endogenously (Fig. 2C). IF of mitotic cells showed that phospho-Aurora B and Skp2 localize similarly mainly at the cellplasm in early mitosis (Fig. 2D). These data further demonstrated the intimate relationship between Skp2 and Aurora B. Skp2 triggers Aurora B ubiquitination As shown that Skp2 interacts with Aurora B and is required for its activation in the process of cell mitosis, we next seek to determine the mechanism how Skp2 regulates Aurora B activation. As aforementioned, Skp2 is known to target protein for ubiquitination, and Aurora B can be ubiquitinated by Cul3/KLHL9/KLHL13 ligase,3 we next want to know whether Skp2 also regulates Aurora B ubiquitination. Indeed, we found that Skp2 remarkably triggered in vivo ubiquitination of Aurora B (Fig. 3A). In further support of this notion, we also did the ubiquitination assay in Skp2 knockdown cells. Compared to the control, the basal level of Aurora B ubiquitination decreased in Skp2 knockdown cells (Fig. 3B). Moreover, we also detected endogenous Aurora B ubiquitination in Hela cells, while the
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Figure 2. Skp2 and Aurora B interact with each other. (A) 293T cells were transfected with XpSkp2 and/or HA-Aurora B, and cells were harvested for coimmmunoprecipitation assay with HA antibody, followed by western blot analysis. (B) 293T cells were transfected with Flag-Skp2 and/or HAAurora B, and cells were harvested for coimmmunoprecipitation assay with Flag antibody, followed by western blot analysis. (C) 293T cells with luciferase control or Skp2 knockdown were harvested and coimmmunoprecipitated with Skp2 antibody, followed by western blot analysis. (D) HeLa cells were transfected with Xp-Skp2, and then were synchronized by nocodazole for 18 hrs, followed by IF analysis with Xpress antibody and phospho-Aurora B antibody.
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Skp2 is essential for cell mitosis progression and spindle checkpoint An earlier study demonstrated that hepatocytes from Skp2¡/¡ mice displayed polyploidy compared to WT mice.14 Here we further confirmed this phenomenon in Skp2¡/¡ MEF cells (Fig. 4A). Since Aurora B is a key kinase in cell mitosis and spindle checkpoint, and our data show that Skp2 is essential for aurora B kinase activity, we next aim to explore the role of Skp2 in cell mitosis and spindle checkpoint. In order to achieve this goal, we first generated stable HeLa cellline with luciferase control and Skp2 knockdown clone #1 and #2 (Fig. 4B). Compared to luciferase control, more cells were arrested in G2/M phase upon Skp2 knockdown after nacodazole treatment (Fig. 4C), also consistent with knockdown efficiency, implying that Skp2 deficiency leads to defect in cell mitosis. In order to further monitor the cell mitotic dynamics, we analyzed the same set of luciferase control and Skp2 knockdown Hela cells with a time-lapse microscopy. We found that it took more time for Skp2 knockdown cells to progress into anaphase compared with control, especially for Skp2 shRNA#1, whose knockdown efficiency was better. For the control cells, the time between nuclear Figure 3. Skp2 triggers Aurora B ubiquitination. (A) 293T cells were transfected with the indicated plasmids envelope breakdown and anaphase and harvested for in vivo ubiquitination assay (see the Materials and Methods for details). (B) 293T cells onset was around 48 mins, while with luciferase control or Skp2 knockdown were transfected with the indicated plasmids and harvested for the time for Skp2 shRNA#1 and in vivo ubiquitination assay. (C) 293T cells with luciferase control or Skp2 knockdown were harvested and shRNA#2 was around 59 and coimmmunoprecipitated with Aurora B antibody, followed by western blot analysis with ubiquitin antibody. (D) 293T cells with Cul-1 or luciferase control knockdown were transfected with indicated plasmids 56 mins independently (Fig. 4E). and harvested for in vivo ubiquitination assay. (E) 293T cells were transfected with HA-Aurora B, Xp-Skp2, As Skp2 interacts with Aurora B along with His-Ub WT, K48R, or K63R mutant and harvested for in vivo ubiquitination assay. and triggers its ubiquitination, we expect that Skp2-mediated cell mitosis progression is Aurora B degradation (Fig. 3A). It indicates that Skp2-induced Aurora B dependent. Therefore we also overexpress Aurora B in Skp2 ubiquitination may be through Lysine (K) 63-linked. Indeed we knockdown clone#1 cells in the Flow Cytometry assay and the found that K63 mutation could impair Aurora B ubiquitination Live-Cell Time-Lapse Imaging experiment. As anticipated, with but Lysine (K) 48 mutation could not (Fig. 3E). Therefore, our Aurora B overexpression, less mitotic cells were arrested in G2/M results suggest that Skp2-mediated Aurora B ubiquitination phase (14.6% vs 28.2%), and the metaphase time came back to may play an important role for the interaction of Skp2 and around 49 mins (Fig. 4D and Fig. 4E), indicating that ectopic Aurora B and the subsequent activation of Aurora B in cell expression of Aurora B in Skp2-depleted cells could partially restore the mitotic defects. Taken together, these data strongly mitosis and spindle checkpoint.
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Figure 4. For figure legend, see page 3882.
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demonstrate the essential role of Skp2 in cell mitosis progression and spindle checkpoint, which is Aurora B dependent.
Discussion CPC is required for correct spindle-kinetochore attachments during chromosome segregation and cytokinesis, of which Aurora B is the key kinase. Deficiency of Aurora B makes the mitotic cells stay at anaphase and can’t proceed to form an organized anaphase spindle and subsequently complete cytokinesis.3 Thus, Aurora B plays a central role in cell mitosis progression and spindle checkpoint. Skp2 is a multifunctional protein. It’s known to play a critical role in cell cycle regulation,6,12 and also demonstrated to contribute to tumorigenesis, cancer development and metastasis in recent years.5,7,15,16 Since Skp2 deficiency results in polyploidy and centrosome overduplication,14 it suggests that Skp2 may be required to maintain genomic stability. Prominently, we have recently demonstrated that Skp2 induced NBS ubiquitination, in turn regulating activation and recruitment of ATM to DNA damage foci in DDR. Furthermore, Skp2 knockdown was shown to exhibit defect in HR repair.8 This important finding further verifies the pivotal role of Skp2 in DDR and maintaining genomic stability. In this study, we provide novel insight into how Skp2 take part in maintaining genomic stability in the process of cell mitosis. We show that Skp2 is required for the activation of Aurora B and its downstream protein, which is the key kinase in cell mitosis and spindle checkpoint. Moreover, Skp2 and Aurora B are shown to interact with each other. Furthermore, Skp2 is also demonstrated to play an essential role in cell mitosis progression and spindle checkpoint, which is Aurora B dependent. Importantly, the E3 ligase activity of Skp2 is required for its role in activating Aurora B. We further demonstrate that Skp2 triggers K63linked ubiquitination of Aurora B. Ubiquitination has multiple important functions including cell cycle control, apoptosis, immune functions, as well as neuron degeneration.17-19 Traditionally, ubiquitination is always targeting protein for degradation. Very recently, protein ubiquitination is also shown to play non-proteolytic functions such as protein trafficking, DNA damage repair as well as protein kinase activation.8,17,20-22 Besides the
role in cell cycle regulation by targeting protein for degradation like p21 and p27, Skp2 is also demonstrated to play central role in DDR and tumorigenesis through kinase activation followed by K63-linked ubiquitination of NBS1 and Akt.7,8 Therefore, in this study Skp2 mediated K63-linked ubiquitination of Aurora B may be responsible for Aurora B kinase activation in cell mitosis and spindle checkpoint. Given that K63-linked ubiquitination is recently shown to provide a docking site for protein-protein interaction,8,17,23 it is very likely that Aurora B ubiquitination may enable the interaction of Skp2 and Aurora B, in turn facilitating activation of Aurora B and its downstream protein. In order to better understand the role of Aurora B ubiquitination, future study will be important to identify the Aurora B ubiquitination sites for Skp2. ATM occupies the central node of the DDR network, thus plays an important role in controlling genomic stability. ATM functions by phosphorylating a list of downstream targets to facilitate cell-cycle checkpoint in response to DDR.24 Interestingly, in the progression of cell mitosis ATM phosphorylates a different but overlapping list of substrates with DDR. For example, some mitotic spindle checkpoint proteins, such as Bub1, Mad1, and Sgo1 are also among the substrates in DDR.25 All these evidence demonstrates that there exists crosstalk between mitotic regulatory machinery and DDR pathway. As is shown recently that Aurora B phosphorylates ATM, and it’s essential for ATM activation in the process of cell mitosis.9 Furthermore, our finding also demonstrates that ATM is a downstream target of Skp2 in the DDR.8 Meanwhile, in this study we found that Aurora B is a novel substrate for Skp2 ubiquitination in cell mitosis. Our study further supports the notion that mitotic checkpoint shares some common molecular components with the DDR pathway. Thus, in order to maintain genomic stability, Skp2 is also involved in the mitotic checkpoint dependent on Aurora B. Given that Skp2 overexpression is a poor prognosis factor for cancer patients and also shown to be critical for the sensitivity of cancer cells to radiotherapy in some cancer cell lines,8,26 Skp2 is recognized as a promising target for cancer treatment. Certain chemical target of Skp2 is also validated to inhibit tumor progression in lung and prostate cancer models.27 Here we show that Skp2 regulates Aurora B activation in the mitosis progression, probably through its ubiquitinating function, and Skp2
Figure 4. (See previous page) Skp2 is essential for cell mitosis progression and spindle checkpoint. (A) Wide Type (WT) and Skp2¡/¡ MEFs were harvested for cell cycle analysis. The left panel shows the representative flow cytometry profile and the right panel displays the statistical results. Error bars represent the standard deviation. (B) Skp2 level was shown for the luciferase control and Skp2 knockdown clone#1 and #2 HeLa cells, which were used for the following FASCS assay (C, D) and Live cell time-lapse imaging assay (E). (C) HeLa cells with luciferase control or Skp2 knockdown were synchronized by nocodazole for 18 hrs and then all the cells were harvested for FASCS assay. The mitotic index was measured based on Histone H3S10p staining. The left panel shows the representative flow cytometry profile and the right panel displays the statistical results. Error bars represent the standard deviation. (D) Skp2 knockdown clone#1 HeLa cells were transfected with vector control or HA-Aurora B plasmids for 30 hrs, synchronized by nocodazole for 18 hrs and then all the cells were harvested for FASCS assay. The mitotic index was measured based on Histone H3S10p staining. The left panel shows the representative flow cytometry profile, the middle panel displays the statistical results, and the right panel shows the Aurora B expression level of the cells used in the assay. Error bars represent the standard deviation. (E) Luciferase control or Skp2 knocking-down HeLa cells expressing H2B-GFP were imaged at every 5 minutes, and Skp2 knockdown clone#1 HeLa cells expressing H2B-GFP transfected with pmCherry-C1-Aurora B was also included. The left panel shows the representative fluorescence video microscopy series from the onset of mitosis to monitor chromosome dynamics and the right panel displays the statistical results. Cell numbers were 100 (Control shRNA), 94(Skp2 shRNA#1), 100(Skp2 shRNA#2), and 72(Skp2 shRNA#1+ Aurora B). Error bars represent the standard deviation.
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deficiency inhibits cell mitosis progression and cytokinesis, which further identifies the function of Skp2 in maintaining genomic stability and subsequently the role in tumorigenesis. Therefore, our data provides new evidence to identify Skp2 as a target for cancer treatment.
were cultured in the medium containing 2mg/ml puromycin for 1 week. The following lentiviral shRNAs were used for transfection: Skp2-lentiviral shRNA#1 (50 -GATAGTGTCATGCTAAAGAAT-3’), Skp2-lentiviral shRNA#2 (50 -GCCTAAGCTAAATCGAGAGAA-3’), Cul1-lentiviral shRNA (50 CCCGCAGCAAATA GTTCATGT-3’).
Materials and Methods
In vivo ubiquitination assay In vivo ubiquitination assay was performed as described.20 In brief, 293T cells were transfected with the indicated plasmids for 48 hrs and harvested by denatured buffer (6 M guanidine-HCl, 0.1 M Na2HPO4/NaH2PO4, 10mMimidazole). The cell extracts were then incubated with nickel beads (Ni-NTA) for 3 hrs, washed, and subjected to western blot analysis.
Cell culture and reagents Mouse embryonic fibroblasts (MEFs) from wild-type and Skp2¡/¡ mice were prepared as previously described.16 293T, HeLa cells were cultured in DMEM containing 10% fetal bovine serum (FBS). To clone Xp-Skp2, Skp2 was amplified by PCR using pcDNA3-Skp2 as a template and inserted into a pcDNA4/ HisMax-TOPO vector (Invitrogen). (His)6-ubiquitin, (His)6ubiquitin K48R, and (His)6-ubiquitin K63R were previously described.20 HA-Aurora B was a gift from Dr. Yi Liang. To clone pmCherry-C1-Aurora B plasmid, Aurora B was amplified by PCR with HA-Aurora B as a template and inserted into the pmCherry-C1 vector. Immunoprecipitation (IP), immunoblotting (IB), and immunofluorescence(IF) IP, IB, and IF were done essentially as described with mild modification.5,8 For IP, cells were lysed by E1A lysis buffer [250 mM NaCl, 50 mM HEPES (pH 7.5), 0.1% NP-40, 5 mM EDTA, protease inhibitor cocktail (Roche)]. The following antibodies were used for IP, IB, and IF: Skp2 antibody (IP: 1:200, IB: 1:2000, Invitrogen), Xpress antibody (IP: 1:500; IB: 1:5000, Invitrogen), Flag antibody (M2) (IP: 1:200; IB: 1:3000, Sigma), Cul-1 antibody (IB: 1:1000, Invitrogen), HA antibody (IP: 1:200; IB: 1:5000, Cell Signal), Aurora B phospho T232 (IF: 1:200; IB: 1:1000, Abcam), phospho-CENP-A (IF: 1:200; IB: 1:1000, Cell Signal). Viral infection For lentiviral short hairpin RNA (shRNA) infection, 293T cells were co-transfected with shRNAs against luciferase or Skp2 along with packing plasmids (deltaVPR8.9) and envelope plasmid (VSVG) using the calcium phosphate precipitation method. Two days after transfection, virus particles containing Skp2 and luciferase shRNAs were used to infect mammalian cells. All the infected cells References 1. Ditchfield C, Johnson VL, Tighe A, Ellston R, Haworth C, Johnson T, Mortlock A, Keen N, Taylor SS. Aurora B couples chromosome alignment with anaphase by targeting BubR1, Mad2, and Cenp-E to kinetochores. J Cell Biol 2003; 161:267-80; PMID:12719470; http://dx.doi.org/10.1083/ jcb.200208091 2. Hauf S, Cole RW, LaTerra S, Zimmer C, Schnapp G, Walter R, Heckel A, van Meel J, Rieder CL, Peters JM. The small molecule Hesperadin reveals a role for Aurora B in correcting kinetochore-microtubule attachment and in maintaining the spindle assembly checkpoint. J Cell Biol 2003; 161:281-94; PMID:12707311; http://dx.doi.org/10.1083/ jcb.200208092
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Flow cytometry For mitotic arrest, Hela cells were synchronized by 200 ng/ml nocodazole for 18 hrs. For flow cytometry analysis, cells were then collected and permeabilized with 0.1% Triton X-100 after fixation in 75% ethanol, stained with Histone H3p antibody (Millipore) and FITC-conjugated second antibody. For DNA content, cells were stained with propidium iodide (PI) (Sigma). After that analysis were performed with a FACS flow cytometer. For Cell-Cycle Analysis, WT and Skp2¡/¡ MEFs cultured in 10% FBS were fixed in 75% ethanol, and stained with PI, followed by flow cytometry analysis. Live-cell time-lapse imaging To monitor chromosome dynamics, GFP-tagged H2B was transfected into HeLa cells with luciferase control or Skp2 knockdown transfected with Aurora B or not. 24 hrs later, cells were imaged at 5min intervals for 24h using live-cell imaging. Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed. Funding
This work was supported by the National Natural Science Foundation of China (81201550), Zhuangjiang Star 2015 of Science and Technology Program of Guangzhou, China (201506010037) to Juan Wu.
3. Sumara I, Quadroni M, Frei C, Olma MH, Sumara G, Ricci R, Peter M. A Cul3-based E3 ligase removes Aurora B from mitotic chromosomes, regulating mitotic progression and completion of cytokinesis in human cells. Dev Cell 2007; 12:887-900; PMID:17543862; http://dx.doi.org/10.1016/j. devcel.2007.03.019 4. Frescas D, Pagano M. Deregulated proteolysis by the Fbox proteins SKP2 and b-TrCP: tipping the scales of cancer. Nat Rev Cancer 2008; 8:438-49; PMID:18500245; http://dx.doi.org/10.1038/nrc2396 5. Lin HK, Wang G, Chen Z, Teruya-Feldstein J, Liu Y, Chan CH, Yang WL, Erdjument-Bromage H, Nakayama KI, Nimer S, et al. Phosphorylation-dependent regulation of cytosolic localization and oncogenic function of Skp2 by Akt/PKB. Nat Cell Biol 2009;
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Volume 14 Issue 24
