Mechanisms controlling the temporal degradation of Nek2A and Kif18A by the APC/C–Cdc20 complex
2013; Springer Nature; Volume: 32; Issue: 2 Linguagem: Inglês
10.1038/emboj.2012.335
ISSN1460-2075
AutoresGarry G. Sedgwick, Daniel Hayward, B. Fiore, Mercedes Pardo, Lu Yu, Jonathon Pines, Jakob Nilsson,
Tópico(s)Genomics and Chromatin Dynamics
ResumoArticle4 January 2013free access Mechanisms controlling the temporal degradation of Nek2A and Kif18A by the APC/C–Cdc20 complex Garry G Sedgwick Garry G Sedgwick The Novo Nordisk Foundation Center for Protein Research, Faculty of Health Science, University of Copenhagen, Copenhagen, Denmark BRIC, University of Copenhagen, Copenhagen, Denmark Search for more papers by this author Daniel G Hayward Daniel G Hayward The Novo Nordisk Foundation Center for Protein Research, Faculty of Health Science, University of Copenhagen, Copenhagen, Denmark BRIC, University of Copenhagen, Copenhagen, Denmark Search for more papers by this author Barbara Di Fiore Barbara Di Fiore The Gurdon Institute and Department of Zoology, University of Cambridge, Cambridge, UK Search for more papers by this author Mercedes Pardo Mercedes Pardo Proteomics Mass Spectrometry Laboratory, The Wellcome Trust Sanger Institute, Cambridge, UK Search for more papers by this author Lu Yu Lu Yu Proteomics Mass Spectrometry Laboratory, The Wellcome Trust Sanger Institute, Cambridge, UK Search for more papers by this author Jonathon Pines Jonathon Pines The Gurdon Institute and Department of Zoology, University of Cambridge, Cambridge, UK Search for more papers by this author Jakob Nilsson Corresponding Author Jakob Nilsson The Novo Nordisk Foundation Center for Protein Research, Faculty of Health Science, University of Copenhagen, Copenhagen, Denmark BRIC, University of Copenhagen, Copenhagen, Denmark Search for more papers by this author Garry G Sedgwick Garry G Sedgwick The Novo Nordisk Foundation Center for Protein Research, Faculty of Health Science, University of Copenhagen, Copenhagen, Denmark BRIC, University of Copenhagen, Copenhagen, Denmark Search for more papers by this author Daniel G Hayward Daniel G Hayward The Novo Nordisk Foundation Center for Protein Research, Faculty of Health Science, University of Copenhagen, Copenhagen, Denmark BRIC, University of Copenhagen, Copenhagen, Denmark Search for more papers by this author Barbara Di Fiore Barbara Di Fiore The Gurdon Institute and Department of Zoology, University of Cambridge, Cambridge, UK Search for more papers by this author Mercedes Pardo Mercedes Pardo Proteomics Mass Spectrometry Laboratory, The Wellcome Trust Sanger Institute, Cambridge, UK Search for more papers by this author Lu Yu Lu Yu Proteomics Mass Spectrometry Laboratory, The Wellcome Trust Sanger Institute, Cambridge, UK Search for more papers by this author Jonathon Pines Jonathon Pines The Gurdon Institute and Department of Zoology, University of Cambridge, Cambridge, UK Search for more papers by this author Jakob Nilsson Corresponding Author Jakob Nilsson The Novo Nordisk Foundation Center for Protein Research, Faculty of Health Science, University of Copenhagen, Copenhagen, Denmark BRIC, University of Copenhagen, Copenhagen, Denmark Search for more papers by this author Author Information Garry G Sedgwick1,2,‡, Daniel G Hayward1,2,‡, Barbara Di Fiore3, Mercedes Pardo4, Lu Yu4, Jonathon Pines3 and Jakob Nilsson 1,2 1The Novo Nordisk Foundation Center for Protein Research, Faculty of Health Science, University of Copenhagen, Copenhagen, Denmark 2BRIC, University of Copenhagen, Copenhagen, Denmark 3The Gurdon Institute and Department of Zoology, University of Cambridge, Cambridge, UK 4Proteomics Mass Spectrometry Laboratory, The Wellcome Trust Sanger Institute, Cambridge, UK ‡These authors contributed equally to this work. *Corresponding author. The Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, Blegdamsvej 3b, Building 6, Copenhagen 2200, Denmark. Tel.:+45 35325053; Fax:+45 35325001; E-mail: [email protected] The EMBO Journal (2013)32:303-314https://doi.org/10.1038/emboj.2012.335 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The Anaphase Promoting Complex/Cyclosome (APC/C) in complex with its co-activator Cdc20 is responsible for targeting proteins for ubiquitin-mediated degradation during mitosis. The activity of APC/C–Cdc20 is inhibited during prometaphase by the Spindle Assembly Checkpoint (SAC) yet certain substrates escape this inhibition. Nek2A degradation during prometaphase depends on direct binding of Nek2A to the APC/C via a C-terminal MR dipeptide but whether this motif alone is sufficient is not clear. Here, we identify Kif18A as a novel APC/C–Cdc20 substrate and show that Kif18A degradation depends on a C-terminal LR motif. However in contrast to Nek2A, Kif18A is not degraded until anaphase showing that additional mechanisms contribute to Nek2A degradation. We find that dimerization via the leucine zipper, in combination with the MR motif, is required for stable Nek2A binding to and ubiquitination by the APC/C. Nek2A and the mitotic checkpoint complex (MCC) have an overlap in APC/C subunit requirements for binding and we propose that Nek2A binds with high affinity to apo-APC/C and is degraded by the pool of Cdc20 that avoids inhibition by the SAC. Introduction The Anaphase Promoting Complex/Cyclosome (APC/C) is a large E3 ubiquitin ligase responsible for targeting proteins for degradation during mitosis. Despite being composed of at least 15 subunits it is absolutely dependent on one of two co-activators, Cdc20 or Cdh1, for its activity (Pines, 2011). The co-activators assist in the binding of substrates to the APC/C and recognize destruction motifs in these, the most common being D boxes or KEN boxes. D-box destruction motifs are bound by a combined interface composed of the co-activator and the APC10 subunit (Carroll and Morgan, 2002; Passmore et al, 2003; Carroll et al, 2005; Matyskiela and Morgan, 2009; Buschhorn et al, 2011; da Fonseca et al, 2011; Schreiber et al, 2011). In addition, the co-activators contain a C box that activates the APC/C (Kimata et al, 2008a) and in combination with a C-terminal IR motif contributes to the binding of co-activators to the APC/C (Passmore et al, 2003; Thornton et al, 2006). The IR motif is able to bind to TPR subunits of the APC/C while the C box is closer to the catalytic core (Wendt et al, 2001; Vodermaier et al, 2003; Matyskiela and Morgan, 2009; Buschhorn et al, 2011; da Fonseca et al, 2011; Schreiber et al, 2011). During mitosis Cdc20 is the essential co-activator that directs the APC/C towards a range of substrates in a defined temporal manner (Pines, 2011). Cdc20 is inhibited by the Spindle Assembly Checkpoint (SAC), a surveillance mechanism ensuring proper chromosome segregation (Musacchio and Salmon, 2007; Nilsson, 2011). The SAC is activated by improperly attached kinetochores and this leads to the binding of the three checkpoint proteins, Mad2, BubR1 and Bub3 to Cdc20, forming the mitotic checkpoint complex (MCC) (Hardwick et al, 2000; Sudakin et al, 2001; Tang et al, 2001; Fang, 2002; Nilsson et al, 2008). The MCC exists either as a free complex or in complex with the APC/C (Sudakin et al, 2001; Morrow et al, 2005; Herzog et al, 2009) via an interaction with the APC8 subunit of the APC/C (Izawa and Pines, 2011). Exactly how Cdc20 is inhibited when in complex with checkpoint proteins is not clear although the BubR1 protein has been proposed to act as a pseudo-substrate, preventing the binding of substrates to Cdc20 (Burton and Solomon, 2007; Lara-Gonzalez et al, 2011). In agreement with this BubR1 interacts with the propeller domain of Cdc20, which would otherwise bind destruction motifs in substrates (Tang et al, 2001; Kraft et al, 2005; Kimata et al, 2008b; Chao et al, 2012). An additional inhibitory mechanism is that Cdc20 becomes a substrate of the APC/C when part of the MCC and this contributes to maintain the SAC (Pan and Chen, 2004; Nilsson et al, 2008). Despite the strong inhibition of Cdc20 by the SAC, certain proteins such as Nek2A and Cyclin A escape this inhibition and are degraded during prometaphase in a Cdc20-dependent manner (Hames et al, 2001; Elzen den and Pines, 2001; Hayes et al, 2006; Wolthuis et al, 2008; Di Fiore and Pines, 2010). Recent work has shown that Cyclin A has high affinity for Cdc20 and can compete with BubR1 for binding to Cdc20 (Di Fiore and Pines, 2010). This property of Cyclin A in combination with direct targeting of the Cyclin A–Cdk1–Cks complex to the APC/C via the Cks subunit allows Cyclin A to be degraded during an active checkpoint (Wolthuis et al, 2008; Di Fiore and Pines, 2010). Nek2A harbours an MR dipeptide at its extreme C terminus that resembles the IR dipeptide found in the co-activators Cdc20 and Cdh1 (Hayes et al, 2006). This MR dipeptide allows Nek2A to bind directly to the APC/C independently of Cdc20 and is required for Nek2A degradation during an active SAC (Hayes et al, 2006). The direct binding of Nek2A bypasses the requirement for the substrate binding propeller domain of Cdc20 and indeed the C-box motif of Cdc20 is sufficient to promote Nek2A degradation (Kimata et al, 2008a). It is however not clear whether the MR dipeptide-mediated interaction with the APC/C is sufficient for degradation during an active SAC, nor whether the MCC and Nek2A can bind simultaneously to the APC/C. Here, we describe the identification of Kif18A as a novel substrate of the APC/C–Cdc20 complex that contains a C-terminal LR dipeptide, which we show is required for binding to the APC/C and for Kif18A degradation. In contrast to Nek2A, Kif18A is not degraded until anaphase, which shows that additional mechanisms contribute to Nek2A degradation during an active SAC. Indeed, we find that C-terminal fragments of Nek2A harbouring the MR motif are not degraded until anaphase and to escape SAC inhibition Nek2A requires dimerization through its leucine zipper. Dimerization of Nek2A is required for stable binding to the APC/C but Nek2A is not able to compete with the MCC and instead binds to the apo form of the APC/C. We propose that stable binding of Nek2A to apo-APC/C is required for efficient degradation by the small amounts of Cdc20 that escape inhibition by the SAC. Results Kif18A binds to the APC/C through a C-terminal LR motif To identify novel APC/C interactors during mitosis, we performed affinity purifications of the complex from HeLa cells using a monoclonal antibody towards the APC4 subunit followed by peptide elution. The APC/C was purified from nocodazole-arrested cells and cells that were released from nocodazole into medium containing the proteasome inhibitor MG132 (Figure 1A). These, and control preparations, were analysed by mass spectrometry. In APC/C purifications from both conditions we identified two peptides of the kinesin Kif18A and in the sample from cells in metaphase we identified 14 peptides of Nek2A. Figure 1.Kif18A binds the APC/C through a C-terminal LR motif. (A) The APC/C complex was affinity purified using an APC4 antibody from nocodazole-arrested cells or cells released from nocodazole into MG132 for 2 h. A fraction of the sample was analysed by silver stain and the remaining sample analysed by mass spectrometry. (B) The amino-acid sequence of the last six amino acids from human Nek2A, Cdc20, Cdh1 and Kif18A is shown. (C) Stable HeLa cell lines expressing FLAG-tagged Kif18A or Kif18AΔLR were arrested with nocodazole and the APC/C complex purified using an APC4 antibody. The binding of Kif18A proteins was analysed by blotting for FLAG. (D) HeLa cells in G2 were injected with plasmids expressing Venus–Kif18A or Venus–Kif18ΔLR fusion proteins and mitotic progression was followed by time-lapse microscopy recording DIC and the Venus signal every 3 min. (E) The total Venus fluorescence was measured and plotted for each construct. At least five cells were analysed for each fusion protein. Download figure Download PowerPoint Inspection of the human Kif18A sequence revealed that the two C-terminal residues were leucine-arginine (LR), which resembles the IR motif and MR motif found in the APC/C co-activators Cdh1, Cdc20 and Nek2A, respectively (Figure 1B). To investigate whether the LR motif in Kif18A had a role in APC/C binding, we created stable HeLa cell lines expressing inducible FLAG-tagged Kif18A or Kif18A where we deleted the last two residues (Kif18AΔLR). From these cell lines arrested in prometaphase we purified the APC/C using the APC4 antibody and monitored Kif18A binding using a FLAG antibody. We found that full-length Kif18A could be co-purified with the APC/C but removal of the C-terminal LR motif greatly reduced the interaction (Figure 1C). To determine when Kif18A is degraded during mitosis we employed a live-cell imaging approach in which Venus-tagged Kif18A constructs were injected into HeLa cells and the total Venus fluorescence used as a read-out of protein stability as cells progress through mitosis (Clute and Pines, 1999). In agreement with characterizations of endogenous Kif18A (Mayr et al, 2007), the protein was stable throughout mitosis but was rapidly degraded at anaphase onset and this depended on its LR motif (Figure 1D and E). To determine whether Kif18A contained additional degradation motifs, we used a similar approach and analysed the degradation of various truncations: Venus-tagged Kif18A 453–898, 653–898 and 800–898. While the Kif18A 453–898 fragment was still degraded at anaphase the two other fragments were stable indicating that the region from 453 to 653 contains motifs required for degradation (Supplementary Figure S1A). Together, these results show that Kif18A binds the APC/C and its degradation at anaphase depends on both a C-terminal LR motif and motifs between residues 453 and 653. Cdc20 is required for Kif18A degradation at anaphase The initiation of Kif18A degradation at anaphase could indicate a specific requirement for the co-activator Cdh1 for Kif18A degradation as Cdh1 can only activate the APC/C when CDK1 activity is low (Kramer et al, 2000). To investigate this, we depleted Cdh1 by RNAi and analysed the impact this had on degradation of Venus–Kif18A stably expressed in U2OS cells. Compared to control treated cells we detected no inhibition of Venus–Kif18A degradation when cells were treated with Cdh1 RNAi (Figure 2A). In a similar approach, cells treated with a control RNAi oligo or a Cdh1 RNAi oligo were released from a nocodazole block and samples collected for western blot analysis at different times following release. Although Cdh1 was efficiently depleted this had no effect on the degradation of Kif18A but did delay the degradation of Aurora A, a Cdh1 substrate (Figure 2B; Floyd et al, 2008; García-Higuera et al, 2008). However, when we depleted Cdc20 and forced cells out of mitosis with the CDK1 inhibitor RO3306 (Vassilev et al, 2006) and monitored Kif18A degradation by western blotting, Kif18A was clearly stabilized (Figure 2C). To determine whether the APC/C–Cdc20 complex was directly able to ubiquitinate Kif18A, we employed an in vitro APC/C ubiquitin ligase assay using in vitro translated Kif18A as a substrate. In these assays, APC/C purified from checkpoint-arrested cells or cells released from the checkpoint using the Aurora B inhibitor ZM447439 (Ditchfield et al, 2003) were used and the Cdc20 that co-purified was responsible for the observed in vitro activity (Supplementary Figure S1B). We found that APC/C–Cdc20 was able to ubiquitinate Kif18A and this was dependent on both the LR motif in Kif18A and sensitive to the SAC (Figure 2D). Figure 2.Kif18A is ubiquitinated by the APC/C–Cdc20 complex. (A) A stable U2OS cell line expressing FLAG–Venus-tagged Kif18A was treated with control RNAi oligo or Cdh1 RNAi oligo and cells were analysed as they progressed through mitosis by time-lapse microscopy recording every 4 min. Total Venus fluorescence was quantified at each time point and the average curve of five cells per condition is shown. (B) Control-depleted or Cdh1-depleted HeLa cells were released from a nocodazole block and samples collected at the indicated time points and analysed for the levels of the indicated proteins by western blot. (C) Control-depleted or Cdc20-depleted HeLa cells were released from a nocodazole block and seeded into fresh medium containing DMSO or 10 μM RO-3306 and samples collected at the indicated time points and analysed by western blot. (D) HeLa cells were treated with taxol (100 ng/ml) for 16 h and 5 μM MG132 was added for 2 h either with or without 3 μM ZM447439 and cells collected by mitotic shake-off. The APC/C was purified using an APC4 antibody and used for in vitro ligase assays where 35S-methionine labelled (35S-met) Kif18A or Kif18A ΔLR were used as substrates. The extent of ligation was determined by autoradiography, high and low exposures are shown. APC4 and BubR1 levels were determined in parallel by western blot analysis of a small fraction of the reaction. Download figure Download PowerPoint We conclude from these data that Kif18A is a novel APC/C–Cdc20 substrate at anaphase. Although it was possible that Kif18A could play a role in localizing the APC/C to the mitotic spindle we did not observe an effect on APC/C spindle localization in Kif18A-depleted cells (Supplementary Figure S2). The leucine zipper of Nek2A is required for degradation during an active SAC Our observation that Kif18A was not degraded until anaphase, despite it showing characteristics similar to Nek2A, raised the question of whether the MR motif in Nek2A was sufficient to target it for degradation during an active checkpoint. Nek2A and Nek2B are splice variants and while Nek2B is stable during prometaphase Nek2A contains an additional terminal exon encoding amino acids 370–445 that allows it to be degraded at nuclear envelope breakdown (NEBD) (Figure 3A and B). To determine whether this region would target Kif18A for degradation during an active checkpoint, we generated a chimeric Kif18A-Nek2A 370–445 fusion and compared it to a construct in which the last 10 amino acids of Kif18A were changed to that of Nek2A. We then created stable isogenic inducible U2OS cell lines that expressed Venus-tagged Kif18A-Nek2A 370–445 and Kif18A-Nek2A 435–445 and used the total fluorescent intensity of the Venus tag as a marker for degradation of these forms of Kif18A. While Kif18A-Nek2A 435–445 was degraded at anaphase Kif18A-Nek2A 370–445 was efficiently degraded at NEBD (Figure 3C and D). Surprisingly when we analysed the degradation of Venus Nek2A 370–445 this was not degraded until anaphase (Figure 3E). Figure 3.The C terminus of Nek2A can target Kif18A for degradation during an active checkpoint. (A) Schematic of Nek2A primary sequence and truncation constructs analysed. (B–E) Stable U2OS/FRT/TRex cell lines expressing the indicated proteins were analysed by time-lapse microscopy and DIC and Venus signal recorded at the intervals indicated. The total fluorescence was determined for each time point and indicated in the plot with reference to the peak intensity, which is set to one. At least five cells in two independent experiments have been analysed for each fusion protein and a representative curve is shown. (B) Venus Nek2A WT (C) Venus Kif18A-Nek2A 370–445 (D) Venus Kif18A Nek2A 435–445, (E) Venus Nek2A 370–445. Download figure Download PowerPoint To understand how Nek2A 370–445 could target a protein for degradation at NEBD despite being an anaphase substrate itself we reinvestigated Nek2A degradation. We created isogenic stable U2OS cells that expressed different inducible Venus–Nek2A constructs. Nek2A is a dimer (Fry et al, 1999) so we made the Venus–Nek2A constructs resistant to RNAi depletion of Nek2A to avoid effects through dimerization with the endogenous protein. Due to the variation in the time it takes to align the chromosomes on the metaphase plate cells spend different times in prometaphase but all the Nek2A constructs analysed below initiated degradation as specified irrespective of how long they spend in prometaphase. In agreement with previous observations, we observed that Venus–Nek2A started to be degraded at NEBD and this depended on the C-terminal MR motif but not a KEN box present in 370–445 (Figures 3B, 4A and B). The Venus–Nek2AΔMR construct was efficiently degraded at anaphase likely due to the presence of additional destruction motifs. When we compared the degradation of Nek2A 370–445, Nek2A 333–445 and Nek2A 301–445 it was clear that including the leucine zipper of Nek2A changed the degradation profile from anaphase to NEBD (Figures 3A, E, 4C and D). To address whether the leucine zipper was required for degradation of Nek2A at NEBD in the context of the full-length protein, we made 15 amino acids deletions in that region and analysed the degradation pattern. In a manner similar to the wild-type protein, Venus Nek2A Δ280–295, with a deletion just outside the leucine zipper, was degraded at NEBD (Supplementary Figure S3) but both Nek2A Δ310–325 and Nek2A Δ325–340 were degraded at anaphase (Figure 4E and F) revealing that an intact leucine zipper is required. Figure 4.Nek2A degradation during prometaphase requires the leucine zipper and the MR motif. (A–F) Stable U2OS/FRT/TRex cell lines expressing the indicated Nek2A proteins were analysed by time-lapse microscopy and DIC and Venus signal recorded at the intervals indicated. The total fluorescence was determined for each time-point and indicated in the plot with reference to the peak intensity, which is set to one. At least five cells in two independent experiments have been analysed for each fusion protein and representative curves are shown. (A) Venus Nek2A KEN/AAA, (B) Venus Nek2A ΔMR (C) Venus Nek2A 333–345 (D) Venus Nek2A 301–445 (E) Venus Nek2A Δ310–325 (F) Venus Nek2A Δ325–340. Download figure Download PowerPoint Our results show that for Nek2A to be degraded during an active checkpoint it needs both the C-terminal MR motif and its leucine zipper. Deletion of either one of these motifs shifts the degradation to anaphase. Dimerization of Nek2A is required for its degradation during an active checkpoint To understand why the leucine zipper was required for Nek2A degradation during an active checkpoint, we analysed the ability of the different Nek2A constructs to bind to the APC/C. Nek2A and Kif18A constructs were transfected into HeLa cells and the APC/C purified from nocodazole-arrested cells (Figure 5A–C). From these experiments, it was clear that both the C-terminal MR motif and leucine zipper of Nek2A were required for high affinity binding to the APC/C, even though the leucine zipper alone did not bind to the APC/C (Figure 5B). Figure 5.Nek2A must dimerize through its leucine zipper to bind to the APC/C when the spindle checkpoint is active. (A) HeLa cells were transiently transfected with 500 ng of the indicated FLAG–Venus–Nek2A or Kif18A fusion expression constructs and arrested in prometaphase by treatment with 200 ng/ml nocodazole for 18 h. The APC/C was immunoprecipitated using anti-APC4 antibodies and blots were probed with a FLAG antibody to assay binding of the specified FLAG–Venus fusion proteins. (B) HeLa cells were transiently transfected with 1 μg of the indicated FLAG–Venus–Nek2A expression constructs, treated with nocodazole for 18 h and then collected by shake-off. The APC/C was immunoprecipitated using anti-APC4 antibodies. Binding of Nek2A proteins was determined by probing with FLAG antibodies and then quantified by Licor. (C) Bar graph comparing binding of the indicated FLAG–Venus–Nek2A proteins to the APC/C after normalization against APC7 and then FLAG inputs. (D) HeLa cells were transiently transfected with increasing amounts of FLAG–Venus–Nek2A WT or L313/L320A expression constructs and treated with nocodazole for 18 h. After APC/C immunoprecipitation with anti-APC4, FLAG–Venus–Nek2A binding was determined using an anti-FLAG antibody. (E) Bar chart comparing relative binding between Nek2A WT and Nek2A L313A/L320A after normalization to APC7 levels. (F) The indicated FLAG–Venus–Nek2A fusion proteins were purified using GFP-Trap beads for 1 h, binding of Myc–Nek2A or the APC/C was determined by Licor using anti-Myc, anti-APC1 and anti-APC7 antibodies. (G) Relative binding of Myc–Nek2A, APC1 and APC7 to FLAG–Venus–Nek2A WT, Nek2A L313A/L320A or Nek2A Δ310–325 after normalization to FLAG–Venus–Nek2A levels. Data are mean±s.d. (n=3) and FLAG–Venus–Nek2A WT is set to 1. (H) Representative degradation curves of Venus Nek2A WT, Venus Nek2A L313A/L320A and Nek2A Δ325–340. Ten cells from two independent experiments were analysed for Nek2A L313A/L320A. Download figure Download PowerPoint As the leucine zipper mediates dimerization of Nek2A we wanted to address whether Nek2A had to be a dimer to bind with high affinity to the APC/C. We mutated two critical leucine residues in the leucine zipper predicted to be required for dimerization (Croasdale et al, 2011), L313A/L320A, and analysed the ability of this mutant to bind the APC/C. At high expression levels, there was still detectable binding of Nek2A L313A/L320A to the APC/C although it was reduced compared to wild type (Figure 5B and C). However, at lower levels of expression Nek2A L313A/L320A was strongly impaired in binding to the APC/C (Figure 5D and E). We also performed purifications of Nek2A, Nek2A L313A/L320A and Nek2A Δ310–325 and analysed the binding both to APC/C and to myc-tagged Nek2A. Again Nek2A L313A/L320A and Nek2A Δ310–325 were strongly impaired in binding to the APC/C and this correlated exactly with the level of binding to myc–Nek2A (Figure 5F and G). When we analysed the degradation pattern of Nek2A L313A/L320A it initiated at NEBD with a slower initial rate than wild-type Nek2A (Figure 5H), suggesting an intermediate phenotype between that of Nek2A and Nek2A Δ310–325/Nek2A Δ325–340. Our results establish that dimerization of Nek2A is critical for high affinity binding to the APC/C and efficient degradation during an active checkpoint. Nek2A binds apo-APC/C during prometaphase There was a clear correlation between the affinity of the different Nek2A constructs for the APC/C and their degradation during an active SAC. As the MCC binds with high affinity to the APC/C, Nek2A might have to compete with the MCC for a common binding site on the APC/C. Potential support for this might be provided by the observation that Nek2A was sensitive to the presence of the MCC in in vitro ubiquitination assays (Supplementary Figure S4A; and see also Herzog et al, 2009). To explore the concept of Nek2A competition with the MCC, we first determined whether Nek2A could bind to the APC/C–MCC complex. We compared the binding of endogenous Nek2A to APC/C purified from HeLa cells treated with MG132 using either an APC4 antibody or to APC/C–MCC purified using a BubR1 antibody. The APC/C purified using an APC/C antibody is an ∼1:1 mixture of apo-APC/C and APC/C–MCC (Herzog et al, 2009). Different amounts of APC/C were analysed by western blotting and it was clear that Nek2A was only present in APC4 purifications, indicating that Nek2A and the MCC did not bind simultaneously to the APC/C (Figure 6A). To determine whether Nek2A was able to displace the MCC from the APC/C, we overexpressed Nek2A more than a 100-fold above its endogenous levels and analysed MCC binding to the APC/C. We only observed a slight decrease in MCC binding upon overexpression of Nek2A in two out of five experiments, indicating that competition with the MCC was an unlikely mechanism by which Nek2A escapes SAC inhibition (Supplementary Figure S4B). To determine whether Nek2A had to be recruited to the APC/C before prometaphase or could also bind once the SAC was activated, we exploited the observation that Nek2A levels increased during a mitotic arrest when the proteasome was blocked with MG132 (Figure 1A). Nocodazole-arrested cells were either treated with DMSO or MG132 and after 2 h the APC/C was purified. This experiment revealed that Nek2A produced during prometaphase could bind to the APC/C (Figure 6B). Taken together, our results clearly suggest that Nek2A binds the apo form of the APC/C during prometaphase. Figure 6.Nek2A binds to the apo-APC/C during prometaphase. (A) APC4 and BubR1 were immunoprecipitated from HeLa cells that were collected by shake-off after treatment with nocodazole (200 ng/ml) for 18 h and 5 μM MG132 for 2 h. Increasing amounts of each IP were loaded. Blots were probed with the indicated antibodies and quantified by Licor. Above each lane, the relative amount of signal is indicated with respect to the maximum signal for APC1, APC4, APC7 and Nek2A. (B) U20S cells were treated for 18 h with nocodazole (200 ng/ml). Mitotic cells were collected by shake-off and incubated in nocodazole ±5 μM MG132 for 2 h and then harvested by shake-off. The APC/C was purified using anti-APC4 antibodies and co-immunoprecipitated endogenous Nek2A was detected using anti-Nek2 antibodies. Download figure Download PowerPoint Nek2A interaction with the APC/C depends on APC8 To gain further insight into how Nek2A interacts with the APC/C during prometaphase to become an efficient substrate, we analysed its interaction with the APC/C. We depleted each of three TPR subunits, APC3, APC6 and APC8, the catalytic APC2 subunit or the substrate binding subunit APC10 and determined the effect on Nek2A binding in APC3 and APC4 purifications (Figure 7A–C). Depletion of any TPR subunit under our conditions, which have a higher ionic strength binding buffer compared to a previous study (Izawa and Pines, 2011), severely impaired binding of both Nek2A and MC
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