Ordered Proteolysis in Anaphase Inactivates Plk1 To

View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by PubMed Central JCBArticle Ordered proteolysis in anaphase inactivates Plk1 to contribute to proper mitotic exit in human cells Catherine Lindon and Jonathon Pines Wellcome Trust/Cancer Research UK Gurdon Institute and Department of Zoology, University of Cambridge, Cambridge CB2 1QR, England, UK e have found that key mitotic regulators show identiﬁed a putative destruction box in Plk1 that is required distinct patterns of degradation during exit from for degradation of Plk1 in anaphase, and have examined W mitosis in human cells. Using a live-cell assay for the effect of nondegradable Plk1 on mitotic exit. Our results proteolysis, we show that two of these regulators, polo-like show that Plk1 proteolysis contributes to the inactivation of kinase 1 (Plk1) and Aurora A, are degraded at different Plk1 in anaphase, and that this is required for the proper times after the anaphase-promoting complex/cyclosome control of mitotic exit and cytokinesis. Our experiments (APC/C) switches from binding Cdc20 to Cdh1. Therefore, reveal a role for APC/C-mediated proteolysis in exit from events in addition to the switch from Cdc20 to Cdh1 control mitosis in human cells. the proteolysis of APC/CCdh1 substrates in vivo. We have Introduction In animal cells, the regulated proteolysis of cyclin A, cyclin APC/C switches from its Cdc20- to Cdh1-activated form, or B1, and securin during mitosis are all essential for the proper whether they are degraded at distinct times, perhaps to coor- timing of events leading up to separation of sister chromatids dinate exit from mitosis. In budding yeast, mitotic exit is under at the onset of anaphase (den Elzen and Pines, 2001; Geley et the tight control of the mitotic exit network (Morgan, 1999), al., 2001; Stemmann et al., 2001; Hagting et al., 2002; a signaling cascade required for the activation of Cdh1 by Leismann and Lehner, 2003). Proteolysis of these key mitotic the Cdc14 phosphatase that can be restrained by a Bub2- regulators is mediated by the anaphase-promoting complex/ dependent checkpoint that monitors the position of the spindle cyclosome (APC/C) ubiquitin ligase and requires the acti- (Li, 1999; Pereira et al., 2000). An equivalent network in vating subunit Cdc20/fizzy (Morgan, 1999; Peters, 2002). mammalian cells has yet to be identified, although homologues The Journal of Cell Biology Cdc20 activity is replaced by that of Cdh1/fizzy-related during of some of the components, such as Cdc14, have been identi- mitotic exit, and the role of Cdh1 in suppressing mitotic fied. In human cells, a homologue of the spindle checkpoint cyclins is essential to establish the G1 phase of the cell cycle protein Mad2 (Mad2B) has been shown to inhibit Cdh1 in (for review see Peters, 2002). However, this switch from vitro (Chen and Fang, 2001), but the role of Mad2B in mitotic Cdc20 to Cdh1 is thought to allow degradation of many exit, if any, is not known. additional substrates because APC/CCdh1 has been shown to Here, we have begun to examine the role and regulation of have broader substrate specificity than APC/CCdc20 (Fang et proteolysis during mitotic exit in mammalian cells, through al., 1998; Pfleger and Kirschner, 2000; Hagting et al., 2002; studying fluorescent protein (FP)–tagged substrates in living Zur and Brandeis, 2002). Amongst the regulators degraded cells. We find that different mitotic regulators are degraded at during mitotic exit in mammalian cells are Cdc20, the polo- different times, indicating that APC/CCdh1 activity may be like kinase 1 (Plk1), the Aurora kinases, and the CENP-E modulated to coordinate mitotic exit and cytokinesis. motor protein (Brown et al., 1994; Weinstein, 1997; Ferris et al., 1998; Honda et al., 2000). However, it has not been shown whether these substrates are all degraded as soon as the Results Initially, we investigated whether it was possible to identify The online version of this article includes supplemental material. differences in the degradation patterns of substrates by ana- Address correspondence to Jonathon Pines, Wellcome Trust/Cancer lyzing immunoblots of endogenous protein levels in HeLa Research UK Gurdon Institute, Tennis Court Rd., Cambridge CB2 1QR, England, UK. Tel.: (44) 1223-334-093. Fax: (44) 1223-334-089. email: Abbreviations used in this paper: APC/C, anaphase-promoting complex/ [email protected] cyclosome; D-box, destruction box; DIC, differential interference contrast; Key words: Aurora; cytokinesis; mitosis; APC/C; Cdh1 FP, fluorescent protein; Plk1, polo-like kinase 1. The Rockefeller University Press, 0021-9525/2004/01/233/9 $8.00 The Journal of Cell Biology, Volume 164, Number 2, January 19, 2004 233–241 http://www.jcb.org/cgi/doi/10.1083/jcb.200309035 233 234 The Journal of Cell Biology | Volume 164, Number 2, 2004 Figure 1. The disappearance of mitotic regulators at exit from mitosis is not concerted. (A and B) HeLa cells were synchronized in prometaphase by a nocodazole block after a thymidine/aphidicolin double block/release (see Materials and methods). Mitotic cells were harvested by shake-off, washed, and replated in the absence of nocodazole. At the time points indicated, samples were processed for immunoblot analysis of the indicated proteins. (A) Parallel samples were processed for analysis of DNA content by flow cytometry, and the M-phase/G1 distribution of each cell population is indicated. Protein loading was assayed by Ponceau S staining and is equivalent for each time point. (B) Extracts were probed with antibodies against p55Cdc20 and Plk1, and with ␤-tubulin as a loading control. We found that the time required for recovery from nocodazole varied between each of the three experiments we performed (compare Plk1 degradation in A and B), but that Cdc20 levels always fell before those of Plk1. ns, extracts from nonsynchronized cells. Note that com- paring levels in unsynchronized cells to mitotic cells in A indicates that PRC1 (but not BubR1) levels are cell cycle regulated. cell extracts. Analysis of extracts from cells synchronized tion from the kinetochores to the midzone of the mitotic through mitotic exit by release from nocodazole-induced ar- spindle (Arnaud et al., 1998; Fig. 2 A, time point 3). The rest showed that the degradation of APC/CCdh1 substrates decline in YFP-Plk1 fluorescence was indeed a result of pro- was not concerted (Fig. 1). Plk1 disappeared from cell ex- teolysis because it was blocked by the proteasome inhibitor tracts earlier than Aurora A and CENP-E, whereas Aurora B MG132 (Fig. 2 B). The onset and rate of YFP-Plk1 degrada- appeared to be degraded later and was still detectable in G1 tion were highly reproducible in this assay (Fig. 2 C), and in cell extracts (Fig. 1 A). In further experiments, p55Cdc20 ap- almost all cells, 60–75% of YFP-Plk1 was degraded during peared to be degraded earlier than Plk1 (Fig. 1 B). PRC1 mitotic exit. Cells expressing very high levels of YFP-Plk1 and BubR1, proposed to be late mitotic substrates (Jiang et were not able to degrade the protein properly and were un- al., 1998; Chan et al., 1999), were not significantly degraded able to exit normally from mitosis (see Fig. 5). Using a U20S after release from nocodazole (Fig. 1 A). Although this anal- cell line stably expressing YFP-Plk1 (Jackman et al., 2003), ysis confirmed that specific mitotic regulators were degraded we have estimated that the usual levels of expression of YFP- during exit from mitosis and indicated that they were de- Plk1 achieved during injection experiments varied from less graded with different kinetics, it did not show exactly when than that of endogenous Plk1 to approximately threefold the degradation of the substrates began. Moreover, we found endogenous level. The timing of YFP-Plk1 degradation was that the rate of recovery from the nocodazole block varied identical in U20S cells (unpublished data). between cells. Therefore, we tagged substrates with FPs to The time at which YFP-Plk1 degradation began appeared use as markers for the endogenous substrates in single-cell to reflect the switch from APC/CCdc20 to APC/CCdh1 at ana- analyses of degradation. phase (Hagting et al., 2002). Therefore, we compared the We began by analyzing Plk1. A GFP-Plk1 chimera had proteolysis of YFP-Plk1 with an in vivo marker for APC/ previously been validated as a marker for the localization of CCdh1. We used a CFP-tagged destruction box (D-box) mu- endogenous human Plk1 (Arnaud et al., 1998), so we exam- tant of securin (db-securin-CFP) that was a substrate for ined whether it was also a suitable marker for the mitotic APC/CCdh1 but not APC/CCdc20 in vitro, and whose degrada- degradation of Plk1. We injected G2 phase HeLa cells with tion in vivo depended on the KEN box motif present in the a cDNA encoding Plk1 tagged at its amino terminus with amino terminus, and on a decline in cyclin B-Cdk1 activity EYFP (YFP-Plk1), and recorded the fluorescence of mitotic (Hagting et al., 2002). db-securin-CFP degradation pre- cells by time-lapse imaging. YFP-Plk1 fluorescence started to ceded that of YFP-Plk1 by several minutes (Fig. 3 A), indi- decline at the beginning of anaphase (Fig. 2), timing that cating that the activation of APC/CCdh1 is not sufficient to was clearly distinguishable from the decrease in cyclin B1- initiate YFP-Plk1 destruction. Our finding that APC/CCdh1 CFP in metaphase (Clute and Pines, 1999; Hagting et al., was active before Plk1 degradation began was consistent 2002; Fig. 2 A; Video 1 and supplemental data, available at with our observation that another APC/CCdh1 substrate, http://www.jcb.org/cgi/content/full/jcb.200309035/DC1).

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