Atypical APC/C‐dependent degradation of Mcl‐1 provides an apoptotic timer during mitotic arrest
2018; Springer Nature; Volume: 37; Issue: 17 Linguagem: Inglês
10.15252/embj.201796831
ISSN1460-2075
AutoresLindsey Allan, Agnieszka Skowyra, Katie I Rogers, Désirée Zeller, Paul R. Clarke,
Tópico(s)Ubiquitin and proteasome pathways
ResumoArticle9 July 2018Open Access Source DataTransparent process Atypical APC/C-dependent degradation of Mcl-1 provides an apoptotic timer during mitotic arrest Lindsey A Allan Lindsey A Allan Division of Cancer Research, Jacqui Wood Cancer Centre, School of Medicine, Ninewells Hospital and Medical School, University of Dundee, Dundee, UK Search for more papers by this author Agnieszka Skowyra Agnieszka Skowyra Division of Cancer Research, Jacqui Wood Cancer Centre, School of Medicine, Ninewells Hospital and Medical School, University of Dundee, Dundee, UK Search for more papers by this author Katie I Rogers Katie I Rogers Division of Cancer Research, Jacqui Wood Cancer Centre, School of Medicine, Ninewells Hospital and Medical School, University of Dundee, Dundee, UK Search for more papers by this author Désirée Zeller Désirée Zeller Division of Cancer Research, Jacqui Wood Cancer Centre, School of Medicine, Ninewells Hospital and Medical School, University of Dundee, Dundee, UK Search for more papers by this author Paul R Clarke Corresponding Author Paul R Clarke [email protected] orcid.org/0000-0003-3525-2622 Division of Cancer Research, Jacqui Wood Cancer Centre, School of Medicine, Ninewells Hospital and Medical School, University of Dundee, Dundee, UK The University of Queensland Diamantina Institute, Faculty of Medicine, Translational Research Institute, Woolloongabba, Qld, Australia Search for more papers by this author Lindsey A Allan Lindsey A Allan Division of Cancer Research, Jacqui Wood Cancer Centre, School of Medicine, Ninewells Hospital and Medical School, University of Dundee, Dundee, UK Search for more papers by this author Agnieszka Skowyra Agnieszka Skowyra Division of Cancer Research, Jacqui Wood Cancer Centre, School of Medicine, Ninewells Hospital and Medical School, University of Dundee, Dundee, UK Search for more papers by this author Katie I Rogers Katie I Rogers Division of Cancer Research, Jacqui Wood Cancer Centre, School of Medicine, Ninewells Hospital and Medical School, University of Dundee, Dundee, UK Search for more papers by this author Désirée Zeller Désirée Zeller Division of Cancer Research, Jacqui Wood Cancer Centre, School of Medicine, Ninewells Hospital and Medical School, University of Dundee, Dundee, UK Search for more papers by this author Paul R Clarke Corresponding Author Paul R Clarke [email protected] orcid.org/0000-0003-3525-2622 Division of Cancer Research, Jacqui Wood Cancer Centre, School of Medicine, Ninewells Hospital and Medical School, University of Dundee, Dundee, UK The University of Queensland Diamantina Institute, Faculty of Medicine, Translational Research Institute, Woolloongabba, Qld, Australia Search for more papers by this author Author Information Lindsey A Allan1,‡, Agnieszka Skowyra1,‡, Katie I Rogers1, Désirée Zeller1 and Paul R Clarke *,1,2 1Division of Cancer Research, Jacqui Wood Cancer Centre, School of Medicine, Ninewells Hospital and Medical School, University of Dundee, Dundee, UK 2The University of Queensland Diamantina Institute, Faculty of Medicine, Translational Research Institute, Woolloongabba, Qld, Australia ‡These authors contributed equally to this work *Corresponding author. Tel: +61 7344 37990; E-mail: [email protected] The EMBO Journal (2018)37:e96831https://doi.org/10.15252/embj.201796831 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 Abstract The initiation of apoptosis in response to the disruption of mitosis provides surveillance against chromosome instability. Here, we show that proteolytic destruction of the key regulator Mcl-1 during an extended mitosis requires the anaphase-promoting complex or cyclosome (APC/C) and is independent of another ubiquitin E3 ligase, SCFFbw7. Using live-cell imaging, we show that the loss of Mcl-1 during mitosis is dependent on a D box motif found in other APC/C substrates, while an isoleucine-arginine (IR) C-terminal tail regulates the manner in which Mcl-1 engages with the APC/C, converting Mcl-1 from a Cdc20-dependent and checkpoint-controlled substrate to one that is degraded independently of checkpoint strength. This mechanism ensures a relatively slow but steady rate of Mcl-1 degradation during mitosis and avoids its catastrophic destruction when the mitotic checkpoint is satisfied, providing an apoptotic timer that can distinguish a prolonged mitotic delay from normal mitosis. Importantly, we also show that inhibition of Cdc20 promotes mitotic cell death more effectively than loss of APC/C activity through differential effects on Mcl-1 degradation, providing an improved strategy to kill cancer cells. Synopsis The APC/C ubiquitin ligase mediates mitotic degradation of the anti-apoptotic regulator Mcl-1 independent of its activator Cdc20. This atypical mechanism provides a timer for cell death induction upon prolonged mitotic arrest, and an opportunity to kill cancer cells in combination with drugs that cause such an arrest. Live-cell imaging shows that mitotic Mcl-1 destruction depends on the APC/C-targeting D box motif. A C-terminal APC/C-interaction IR tail converts Mcl-1 into a Cdc20- and checkpoint-independent substrate. Constitutive slow Mcl-1 degradation ensures cell death induction only after prolonged mitotic delay but not during normal mitosis. Specific inhibition of Cdc20-dependent APC/C functions enhances mitotic cell death through differential effects on Mcl-1 and cyclin B stabilities. Introduction Cellular responses to the disruption of mitosis include the induction of cell death by apoptosis, which normally prevents the propagation of chromosomal abnormalities that result from defects in mitotic spindle assembly. Identification of the mechanisms controlling the induction of apoptosis during mitosis is not only important to understand how chromosome instability is generated in cancer, but also how cells respond to anti-cancer chemotherapeutics such as microtubule poisons that target the spindle and cause an arrest or prolonged delay in mitosis. The timing of exit from mitosis is determined by the mitotic or spindle assembly checkpoint. During prometaphase, kinetochores that are unattached to spindle microtubules signal through the Mps1-dependent generation of the mitotic checkpoint complex (MCC), which inhibits the Cdc20-dependent activation of the anaphase-promoting complex or cyclosome (APC/C), an E3 ligase that ubiquitinates key substrates and targets them for destruction by the proteasome (Pines, 2011; Sivakumar & Gorbsky, 2015). If the proper bi-allelic attachment of microtubules to kinetochores fails or is prevented pharmacologically, for instance by a drug that disrupts microtubule dynamics, then a cell is held in mitosis for a prolonged period by sustained activation of the checkpoint. A key substrate of APC/CCdc20 is cyclin B1, the regulatory subunit of the CDK1-cyclin B1 protein kinase that acts as the master regulator of mitosis. During a prolonged mitotic arrest, the loss of cyclin B1 is restrained, although it eventually drops below the threshold required to maintain the arrest despite the checkpoint (Brito & Rieder, 2006). The propensity of cells to undergo apoptosis correlates with the duration of mitosis (Bekier et al, 2009; Huang et al, 2009; Colin et al, 2015), which indicates a progressive increase in pro-apoptotic signalling until it reaches a threshold sufficient to initiate cell death. Whether or not a cell dies in mitosis depends upon whether the apoptotic threshold is breached before exit from mitosis (Gascoigne & Taylor, 2008). Entry into interphase is generally associated with increased cell survival, probably due to a raised apoptotic threshold, although cells that have been arrested for a prolonged period in mitosis can subsequently undergo cell cycle arrest or post-mitotic cell death (Bekier et al, 2009; Huang et al, 2009; Uetake & Sluder, 2010; Colin et al, 2015). Mcl-1 has emerged as a major determinant of the cellular response to microtubule poisons that target mitotic cells (Harley et al, 2010; Shi et al, 2011; Wertz et al, 2011; Dikovskaya et al, 2015; Haschka et al, 2015; Topham et al, 2015; Sloss et al, 2016). Mcl-1 is a member of the anti-apoptotic Bcl-2 family of proteins that suppress the activation of caspases, which are apoptotic proteases that bring about cellular destruction (Budihardjo et al, 1999). Antagonism of anti-apoptotic Bcl-2 family proteins, which are often over-expressed in tumours, has been proposed as a valuable anti-cancer strategy (Opferman, 2016). Importantly, Mcl-1 is degraded by an ubiquitin-proteasome-dependent mechanism in response to the disruption of mitosis (Harley et al, 2010; Wertz et al, 2011). Removal of Mcl-1 promotes mitotic cell death while stabilisation of the protein inhibits apoptosis induced by mitotic arrest (Colin et al, 2015; Topham et al, 2015; Sloss et al, 2016). In addition, Mcl-1 controls cell fate through suppression of both caspase-dependent activation of DNA damage signalling at telomeres during a prolonged mitosis and the subsequent activation of p53 in cells that initially survive mitotic arrest and enter interphase (Colin et al, 2015; Hain et al, 2016). Mcl-1 is therefore a key modulator of effects of mitotic disruption, and the mechanisms that determine Mcl-1 instability during mitotic arrest are crucial to understand cellular responses to anti-mitotic drugs. The ubiquitin E3 ligase that catalyses the poly-ubiquitylation of Mcl-1 and primes it for proteasomal destruction during mitosis is therefore of significant interest. Previous biochemical experiments have suggested roles for SCFFbw7 (Wertz et al, 2011) and APC/CCdc20 (Harley et al, 2010) in the control of Mcl-1 stability during mitosis, although Fbw7 might have an indirect role through control of the progression of mitosis (Finkin et al, 2008) and the requirement for Cdc20 for mitotic Mcl-1 destruction has been questioned (Díaz-Martínez et al, 2014; Sloss et al, 2016). In this report, we establish by live-cell imaging that the loss of Mcl-1 during mitotic arrest requires APC/C and not SCFFbw7. We show that Mcl-1 is an atypical APC/C substrate with an unusual dependency on Cdc20. A C-terminal isoleucine-arginine (IR) motif makes recognition of Mcl-1 by APC/C insensitive to the level of Cdc20. This mechanism ensures that Mcl-1 is degraded during a prolonged mitosis at a relatively slow rate that is not controlled by the spindle assembly checkpoint. Mcl-1 destruction is not accelerated when the checkpoint is relieved, preventing catastrophic apoptosis as a cell progresses out of mitosis. We propose that the steady rate of Mcl-1 loss during mitotic arrest enables it to act as a timer that measures the period of mitotic arrest independently of checkpoint strength and thereby distinguishes severe mitotic disruption from the normal progression of mitosis. Consistent with an atypical mechanism of APC/C-mediated Mcl-1 destruction, we show that distinct means of inducing mitotic arrest have differential effects on Mcl-1 degradation compared to that of cyclin B1, yielding alternative cell fate profiles. These properties provide opportunities to enhance cell killing in response to chemotherapeutic drugs that target mitotic cells, particularly in cancer cells that are prone to mitotic slippage. Thus, we show that the propensity to undergo mitotic cell death is determined by both the temporal period of mitosis and the nature of the mitotic arrest. Results Mcl-1 is targeted for degradation during mitotic arrest by APC/C Previous work has identified two ubiquitin E3 ligases, namely APC/C and SCFFbw7, as determinants of Mcl-1 loss in response to microtubule poisons (Harley et al, 2010; Wertz et al, 2011). To test the relative contributions of these enzymes to Mcl-1 degradation specifically during mitotic arrest, we analysed by Western blotting the requirement for each E3 ligase in pools of isolated rounded-up mitotic cells synchronised in the period of mitotic arrest by treatment with the microtubule poison nocodazole (830 nM). siRNA-mediated knockdown of APC11, a catalytic subunit of APC/C, stabilised Mcl-1 in HeLa cells during a synchronised mitotic arrest (Fig 1A). Although knockdown of Fbw7 (encoded by the FBXW7 gene and also known as hCDC4; Davis et al, 2014) elevated Mcl-1 protein levels in interphase, it did not prevent Mcl-1 degradation during mitotic arrest (Fig 1A). While Mcl-1 degradation did appear to be slightly reduced in DLD1 FBW7−/− knockout cells treated with 100 nM paclitaxel (Fig EV1A and B), the mitotic arrest in these cells was compromised compared with wild-type cells as demonstrated by lower levels of both MPM2 and phosphorylated APC3 and by the reduced number of cells positive for histone-H3 phosphorylated on Ser10 (p-Ser10-H3), consistent with previous data (Finkin et al, 2008; Wertz et al, 2011). Importantly, when a higher concentration of paclitaxel (500 nM) was used to better maintain mitotic arrest in DLD1 FBW7−/− cells (Fig EV1C), Mcl-1 degradation occurred as in wild-type cells (Fig 1B). As observed in HeLa cells, knockdown of APC11 also stabilised Mcl-1 in mitotically arrested DLD1 WT cells compared to control knockdown cells (siLuc) (Fig 1C). These results indicate that Mcl-1 degradation during mitotic arrest is mediated by the APC/C, whereas loss of Fbw7 causes an apparent stabilisation of Mcl-1 because of its ability to promote premature exit from mitosis rather than by inhibiting Mcl-1 degradation during mitotic arrest. Figure 1. Mcl-1 destruction during mitotic arrest is mediated by the APC/C, not SCFFbw7 HeLa cells transfected with control (siLuc), APC11 or Fbw7 siRNA for 48 h were synchronised in mitotic arrest with 830 nM nocodazole for 2 and 6 h and samples analysed with antibodies as indicated. An untreated sample (−) was used as a control. DLD1 FBW7+/+ (WT) and FBW7−/− (KO) cells were released from a double thymidine block prior to addition of paclitaxel (500 nM). Samples were collected at times indicated after thymidine washout. DLD1 cells WT for FBW7 transfected with control (siLuc) or APC11 siRNA were treated as in (B). HeLa-YFP-Mcl-1 cells were transfected with siRNA as indicated and treated with nocodazole 48 h later. YFP-Mcl-1 fluorescence was monitored from the time of mitotic entry. Error bars represent ±SD, n = 3. Scale bar, 10 μm. Source data are available online for this figure. Source Data for Figure 1 [embj201796831-sup-0002-SDataFig1.pdf] Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Loss of FBW7 does not prevent Mcl-1 degradation during mitosis but does compromise mitotic arrest (related to Fig 1) Characterisation by Western blotting of DLD1 FBW7+/+ (WT) and FBW7−/− (KO) cells. *denotes a non-specific band. Elevation of Myc, a substrate of Fbw7, demonstrates the effect of Fbw7 loss. Western blotting analysis of Mcl-1 degradation in DLD1 WT and FBW7 KO in cells arrested with 100 nM paclitaxel (upper panels). The efficacy of 100 nM paclitaxel to induction of mitotic arrest is indicated by flow cytometric analysis of histone H3 phosphorylated on Ser10 (lower panel). Analysis of mitotic arrest induced by treatment of DLD1 WT and FBW7 KO cells with 500 nM paclitaxel as in (B). YFP-Mcl-1 is degraded as endogenous Mcl-1 during a synchronised mitotic arrest. Live-cell imaging of the degradation of cyclin A-Venus and YFP-Nek2A during mitotic arrest induced by treatment with 830 nM nocodazole. Error bars represent ±SD, n = 3. Confirmation of siRNA knockdown of Fbw7 or APC2 and APC11 and the stabilisation of cyclin E and Myc following Fbw7 depletion in HeLa cells stably expressing YFP-Mcl-1. *denotes a non-specific band. Source data are available online for this figure. Download figure Download PowerPoint To analyse rates of protein destruction more accurately and exclusively in mitotic cells with precise correlation to the timing of mitosis, we established cell lines expressing inducible YFP-tagged Mcl-1 and used live-cell imaging of the fluorescent fusion protein to monitor events in individual mitotic cells. YFP-Mcl-1 was degraded during mitotic arrest similar to endogenous Mcl-1 (Fig EV1D). However, compared with some other APC/C substrates, such as Nek2A and cyclin A, which are degraded rapidly despite an active checkpoint (Hayes et al, 2006; Di Fiore & Pines, 2010), the rate of YFP-Mcl-1 degradation was relatively slow (compare Fig 1D with Fig EV1E), as would be required for an apoptotic timer. Consistent with our previous results, YFP-Mcl-1 degradation in cells arrested in mitosis was inhibited by loss of APC/C activity (concomitant knockdown of the catalytic subunits APC2 and APC11) but was unaffected by depletion of Fbw7 (Fig 1D), even though the efficiency of knockdown was sufficient to stabilise protein levels of cyclin E and c-Myc, both well-documented Fbw7 substrates (Davis et al, 2014; Fig EV1F). Together, these results confirm that Mcl-1 degradation during mitotic arrest is not regulated by Fbw7 but is instead directed by the APC/C. A putative D box is a determinant of Mcl-1 degradation We have identified a putative D box motif in Mcl-1, as well as a C-terminal IR tail, which is also found in the APC/C adaptor/activator proteins Cdc20 and Cdh1 (Harley et al, 2010; Fig 2A). Immediately C-terminal to the putative D box, we also noted a region containing charged residues including putative acceptor lysine residues that form a loosely defined sequence also found in other APC/C substrates. This region has been proposed to be a docking site for UbcH10, the initiating E2 for the APC/C (Williamson et al, 2011; Chang et al, 2015). Mutation of two key residues in the D box strongly inhibited YFP-Mcl-1 degradation (Fig 2B), consistent with our previous Western blotting analysis (Harley et al, 2010). Mutation of charged residues including two lysine residues C-terminal to the D box also stabilised YFP-Mcl-1, although to a lesser extent than the D box mutation (Fig 2C). The spacing between the D box and the lysines contained in this charged region suggests that they may be targeted by UbcH10-APC/C for ubiquitination (Chang et al, 2015). Thus, the partial stabilisation exhibited by this mutant may be due either to loss of an UbcH10 docking site or loss of the optimal ubiquitin acceptor site(s). Nevertheless, these results attest further to the identity of the APC/C as an E3 ubiquitin ligase that targets Mcl-1 during mitotic arrest. Figure 2. The C-terminal IR motif is dispensable for Mcl-1 degradation A. Sequence alignment of D box and IR motifs in Mcl-1 and other APC/C substrates. Key residues are indicated in the D box (blue) and C-terminal to the D box (red); those mutated in this study are underlined. The IR motif at the C-terminus is highlighted (italics). B–D. Degradation of WT YFP-Mcl-1 protein compared with a D box mutant (B), charged residue mutant (C) and IR-deleted (ΔIR) Mcl-1 proteins (D) during mitotic arrest by time-lapse imaging. Error bars represent ±SD, n = 3. Scale bar, 10 μm. E. U2OS cells expressing Flag-tagged WT, D box mutant or D box mutant ΔIR Mcl-1 proteins were synchronised in mitotic arrest with paclitaxel (100 nM). Samples were collected at times indicated and blotted with antibodies as shown. Source data are available online for this figure. Source Data for Figure 2 [embj201796831-sup-0003-SDataFig2.pdf] Download figure Download PowerPoint Deletion of the C-terminal IR motif (ΔIR) had no impact on the rate of YFP-Mcl-1 loss during mitotic arrest (Fig 2D), in contrast to Nek2A where a C-terminal methionine-arginine (MR) motif contributes to maximal Nek2A degradation during mitosis (Hayes et al, 2006). However, expression of the Mcl-1 D box mutant stabilised both cyclin A and cyclin B1 during mitotic arrest, as previously noted (Harley et al, 2010), and this was also the case for Nek2A degradation (Fig 2E), suggesting a dominant inhibitory effect on the APC/C. Remarkably, the ability of the Mcl-1 D box mutant protein to stabilise other substrates of the APC/C was lost when the IR motif was also deleted (DboxΔIR). Thus, although it is not necessary for Mcl-1 degradation, the IR motif appears to regulate the manner in which Mcl-1 engages with the APC/C to the exclusion of other substrates when the interaction is stabilised. The IR tail of Mcl-1 influences APC/C subunit requirements The APC/C is a multisubunit enzyme that is activated by a co-factor, generally Cdc20 during mitosis and Cdh1 following mitotic exit. APC2 and APC11 form the catalytic core of APC/C while APC10 together with Cdc20 forms a receptor that recognises and binds to the D box of the substrate. Cdc20 and Cdh1, like Mcl-1, possess a C-terminal IR motif through which they bind to the scaffolding subunit, APC3. This is required for the substrate recruitment function of Cdc20/Cdh1. Cdc20 also binds to another scaffold subunit, APC8, to elevate the activity of APC/C (Chang et al, 2014, 2015). To examine the role of the IR motif in Mcl-1, we investigated the requirement for individual components of the APC/C in the degradation of wild-type (WT) and IR-deleted (ΔIR) YFP-Mcl-1 during a nocodazole arrest. Knockdown of either APC3 or APC11 stabilised YFP-Mcl-1 (WT Mcl-1; Figs 3A and EV2A). Similarly, YFP-Mcl-1ΔIR was stabilised by loss of APC11; however, unlike the wild-type protein, its degradation was hardly affected by APC3 knockdown (Figs 3B and EV2A). This suggests that the IR motif of Mcl-1 interacts with APC3 in a manner analogous to the IR tail of Cdc20 (Chang et al, 2014) and as suggested for the MR of Nek2A (Hayes et al, 2006), thus aiding recruitment of the protein to the APC/C. Interestingly, depletion of APC8 had no effect on the degradation of either protein (Fig 3A and B), which is consistent with the lack of stimulation by Cdc20 under these conditions. In contrast, and as expected, both cyclin A and cyclin B1 were stabilised by knockdown of each of the three APC/C subunits tested (Fig EV2B–D). Figure 3. The IR tail alters APC/C subunit requirements for Mcl-1 degradation A, B. APC3, APC8 or APC11 were knocked down by siRNA prior to treatment with nocodazole to induce mitotic arrest. The effect on the degradation of YFP-Mcl-1 WT (A) and ΔIR (B) protein was analysed by time-lapse microscopy. Representative images (left) and quantification (right) are shown. Error bars represent ±SD, n = 3. Scale bar, 10 μm. C, D. The effect of knocking down APC10 on YFP-Mcl-1WT (C) and ΔIR (D) proteins during mitotic arrest is shown as for (A, B). Error bars represent ±SD, n = 3. Scale bar, 10 μm. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. APC/C subunit requirements for Mcl-1 degradation during mitotic arrest (related to Fig 3) A, B. Confirmation of siRNA knockdown of APC3, APC8 and APC11 in HeLa cells stably expressing (A) YFP-Mcl-1 or YFP-Mcl-1ΔIR or (B) cyclin A-Venus or cyclin B1-Venus. C, D. APC3, APC8 or APC11 were knocked down by siRNA prior to treatment with nocodazole to induce mitotic arrest. The effect on the degradation of cyclin A-Venus protein in 830 nM nocodazole (C) and cyclin B1-Venus protein in 33 nM nocodazole (D) was analysed by time-lapse microscopy. The nocodazole concentration was reduced to analyse cyclin B1 degradation since cyclin B1 was stable for the duration of the experiment at a higher nocodazole concentration (830 nM, data not shown). Representative images (left) and quantification (right) are shown. Error bars represent ±SD, n = 3. Scale bar, 10 μm. E. Confirmation of siRNA knockdown of APC10 in HeLa cells stably expressing YFP-Mcl-1 WT or ΔIR, or cyclin B1-Venus. F. APC10 was knocked down by siRNA prior to treatment with nocodazole (33 nM) to induce mitotic arrest. The effect on the degradation of cyclin B1-Venus was analysed by time-lapse microscopy. Error bars represent ±SD, n = 3. Source data are available online for this figure. Download figure Download PowerPoint Interestingly, whereas degradation of WT YFP-Mcl-1 was insensitive, like cyclin A (Izawa & Pines, 2011), to loss of APC10 (Fig 3C), the degradation of YFP-Mcl-1ΔIR was retarded to an extent similar to that observed for cyclin B1 (Figs 3D, and EV2E and F). This suggests that APC10 is more important for Mcl-1ΔIR recruitment because loss of the IR-APC3 module invokes a greater reliance on the D box-APC10 interaction. Together, the differences in APC/C subunit requirement between WT Mcl-1 and Mcl-1ΔIR destruction indicate that the IR tail alters the nature of the interaction between Mcl-1 and the APC/C. The IR tail makes Mcl-1 degradation insensitive to the mitotic checkpoint Although deletion of the IR motif had no discernible effect on YFP-Mcl-1 degradation during a strong mitotic arrest (Fig 2D), it did affect the rate of degradation upon exit from an unperturbed mitosis (Fig 4A). Fluorescence intensity declined sharply in YFP-Mcl-1ΔIR-expressing cells as they progressed through mitosis and continued as the cells underwent cytokinesis, whereas YFP-Mcl-1 declined more slowly and then became stabilised. IR-deleted Mcl-1 was, therefore, degraded more rapidly and completely than wild-type Mcl-1 protein when the checkpoint was switched off. Figure 4. The IR tail renders Mcl-1 degradation insensitive to the mitotic checkpoint A. Representative images (left panels) and quantification (right panels) of the degradation of YFP-Mcl-1 WT and ΔIR proteins in unperturbed mitosis. Fluorescence (upper panels) and transmitted light (lower panels) images are shown. Scale bar, 10 μm. B, C. Representative images (left panels) and quantification (right panels) of the degradation of YFP-Mcl-1 WT (B) and ΔIR (C) in cells entering mitosis in the presence of nocodazole and indicated concentration of reversine. In (B), mean values at each time point obtained from three experiments are shown. Error bars represent ±SD. In (C), quantification of 10 individual cells from one representative experiment is shown (right). The traces are overlaid with mean values ±SD (open characters, n = 3). Scale bar, 10 μm. Download figure Download PowerPoint To investigate this further, we analysed the effect on Mcl-1 stability of inhibiting the checkpoint in cells arrested in mitosis by nocodazole. Under these conditions, the degradation of YFP-Mcl-1 was unaffected when the checkpoint was weakened by the Mps1 kinase inhibitor, reversine (Santaguida et al, 2010). Even in cells treated with a high dose of reversine, which caused them to exit mitosis rapidly, Mcl-1 degradation proceeded at a steady rate (Fig 4B). In stark contrast, the rate of YFP-Mcl-1ΔIR degradation increased dramatically as the checkpoint was weakened by increasing concentrations of reversine (Fig 4C). Thus, the APC/C-dependent degradation of Mcl-1 during mitosis is held in check by the IR tail. Without the interaction of the IR tail with the APC/C, Mcl-1 undergoes very rapid degradation when the checkpoint is switched off. The IR tail converts Mcl-1 from a Cdc20-dependent substrate to one that is degraded relatively slowly and independently of checkpoint control Our finding that Mcl-1 degradation is unaffected by the spindle assembly checkpoint is unusual for an APC/C substrate, since the rates of loss of even those substrates such as cyclin A that are rapidly degraded by a Cdc20-dependent mechanism during mitotic arrest are still influenced by the checkpoint (Collin et al, 2013). We found that when Cdc20 was efficiently depleted by siRNA-mediated knockdown (Fig EV3A and B), YFP-Mcl-1 degradation and ubiquitination were not inhibited (WT Mcl-1; Figs 5A and EV3C). Rather there was a slight acceleration of YFP-Mcl-1 degradation when Cdc20 was removed. These results are in contrast to our previous biochemical experiments in which Cdc20 was depleted, albeit inefficiently (Harley et al, 2010), but in agreement with the lack of stabilisation of Mcl-1 following Cdc20 knockdown observed by others (Díaz-Martínez et al, 2014; Sloss et al, 2016). Indeed, we did not observe a requirement for an APC/C co-factor for Mcl-1 degradation since even efficient co-depletion of both Cdc20 and Cdh1 failed to inhibit the loss of YFP-Mcl-1 during mitotic arrest (Fig EV3D). In contrast, concurrent knockdown of APC2 and APC11 both prevented YFP-Mcl-1 ubiquitination (Fig EV3C) and stabilised the protein (Fig 1D). Similarly, under stringent checkpoint conditions, YFP-Mcl-1ΔIR degradation was also unaffected by knockdown of Cdc20 (Figs 5B and EV3E), despite the reduction in Cdc20 being sufficient to stabilise cyclin A (Fig EV3F). Click here to expand this figure. Figure EV3. Cdc20 and Cdh1 are not required for the degradation of Mcl-1 during mitotic arrest (related to Fig 5) Confirmation of siRNA knockdown of Cdc20 in HeLa cells stably expressing YFP-Mcl-1 WT. Western blotting of cells synchronised during mitotic arrest with nocodazole (830 nM) confirmed that the degradation of YFP-Mcl-1 protein reflected temporally that of endogenous Mcl-1 and that both were unaffected by Cdc20 depletion. Mcl-1 ubiquitination is inhibited by depletion of the catalytic APC/C subunits but not Cdc20. HeLa-YFP-Mcl-1 cells transfected with His-ubiquitin were depleted of APC2 and APC11 or Cdc20. Dox was added where indicated to induce expression of YFP-Mcl-1. Ubiquitinated proteins were purified and samples blotted for Mcl-1. Cell lysates (input) were also blotted with indicated antibodies to demonstrate knockdown of protein expre
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