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RCS1, a Substrate of APC/C, Controls the Metaphase to Anaphase Transition

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RCS1, a Substrate of APC/C, Controls the Metaphase to Anaphase Transition RCS1, a substrate of APC/C, controls the metaphase to anaphase transition Wei-meng Zhao*, Judith A. Coppinger†, Akiko Seki*, Xiao-li Cheng*, John R. Yates III†, and Guowei Fang*‡ *Department of Biological Sciences, Stanford University, Stanford, CA 94305-5020; and †Department of Chemical Physiology, The Scripps Research Institute, La Jolla, CA 92037 Edited by Stephen J. Elledge, Harvard Medical School, Boston, MA, and approved June 26, 2008 (received for review September 28, 2007) The anaphase-promoting complex/cyclosome (APC/C) controls the ing of its function in the cell cycle. We report here the identification onset of anaphase by targeting securin for destruction. We report of a substrate of APC/C through a powerful functional genomic here the identification and characterization of a substrate of APC/C, analysis (5) and the characterization of the physiological function of RCS1, as a mitotic regulator that controls the metaphase-to-anaphase this substrate at the metaphase-to-anaphase transition. transition. We showed that the levels of RCS1 fluctuate in the cell cycle, peaking in mitosis and dropping drastically as cells exit into G1. Results Indeed, RCS1 is efficiently ubiquitinated by APC/C in vitro and de- Functional Genomic Analyses Identified Substrates of APC/C. To graded during mitotic exit in a Cdh1-dependent manner in vivo. develop a systematic approach for the identification of APC/C APC/C recognizes a unique D-box at the N terminus of RCS1, as substrates (5), we analyzed 16 known substrates of the APC/C that mutations of this D-box abolished ubiquitination in vitro and stabi- have been characterized at the onset of this study. These substrates lized the mutant protein in vivo. RCS1 controls the timing of the included cyclin A, cyclin B, Plk1, Aurora A, Aurora B, Nek2, Cdc20, anaphase onset, because the loss of RCS1 resulted in a faster pro- UbcH10, securin, GTSE1, anillin, TPX2, Cdc6, geminin, ribonu- gression from the metaphase to anaphase and accelerated degrada- cleotide reductase M2, and SnoN (1–4, 7, 8). The first 15 substrates tion of securin and cyclin B. Biochemically, mitotic RCS1 associates are cell-cycle regulators (1), whereas the last one, SnoN, acts in cell with the NuRD chromatin-remodeling complex, and this RCS1 com- signaling (7). Genes involved in cell cycle regulation have been plex is likely involved in regulating gene expression or chromatin shown to covary transcriptionally during tumorigenesis, as these structure, which in turn may control anaphase onset. Our study regulators are under selective pressures to act coordinately as one uncovers a complex regulatory network for the metaphase-to- module for cell proliferation (9, 10). Indeed, the expression of the anaphase transition. first 15 cell-cycle substrates of the APC/C covaries transcriptionally across hundreds of tumor tissues/cell lines (10), whereas the ex- HDAC1/2 ͉ mitosis ͉ ubiquitin-dependent proteolysis ͉ Cdh1 pression of SnoN does not (9, 10). Furthermore, of the 15 cell cycle substrates, the first 12 act in mitosis and cytokinesis (1–4, 7), and all biquitin-mediated proteolysis regulates key transitions in bi- 12 genes are transcriptionally induced in G2 or in mitosis (11). We Uology. For example, the anaphase-promoting complex/ concluded that a common theme for cell cycle substrates is their cyclosome (APC/C) is an essential ubiquitin ligase that controls the transcriptional covariation with each other across hundreds of ordered degradation of over 20 cell-cycle regulators in mitosis and tumor tissues. Substrates that only function in mitosis and cytoki- nesis are likely to be induced in G2 or in mitosis. Thus, we searched G1 (1). Degradation of securin by the APC/C at the onset of anaphase allows activation of separase and cleavage of the cohesin published microarray databases for genes that fulfill these two complex, leading to segregation of sister chromatids. Similarly, criteria (10, 11) and then tested the corresponding proteins for their APC/C-dependent destruction of cyclin B at anaphase inactivates ability to be ubiquitinated by the active APC/C-Cdh1 in a recon- the cyclin B/Cdk1 kinase and results in mitotic exit. At telophase stituted assay (4, 12). From these experiments, we identified eight and cytokinesis, APC/C controls the degradation of a group of new substrates of the APC/C: ECT2, KIF2C/MCAK, KIFC1/ cell-cycle proteins, such as Aurora A/B, Plk1, TPX2, CKAP2 and HSET, MELK, MKLP2, Par-1a/MARK3, and two proteins, anillin (2–5), to ensure the ordered mitotic exit and cytokinesis. FLJ20354 and FLJ10156, all of which are efficiently ubiquitinated by the APC/C in a Cdh1-dependent manner [Fig. 1 and supporting Beyond mitosis, APC/C is also involved in the G1/S transition and DNA replication. information (SI) Fig. S1]. We named FLJ10156 as a regulator of The ordered destruction of APC/C substrates is at least in part chromosome segregation (RCS1) and reported here the charac- controlled by the association of APC/C with its regulators, Cdc20 terization of FLJ10156 as a substrate of APC/C that controls the and Cdh1 (1). Cdc20 binds to APC/C in mitosis and activates its metaphase-to-anaphase transition. ligase activity at the anaphase onset to trigger the degradation of securin, cyclin B, and Xkid, whereas Cdh1 is required for the RCS1 Is a Substrate of the APC/C in Vitro. Two activators, Cdc20 and Cdh1, control the activity of APC/C in anaphase and from activation of APC/C during mitotic exit and in G1. Cdc20 and Cdh1 recognize two motifs in the substrates: the destruction box (D-box; mitotic exit to G1, respectively (1, 7). In vitro ubiquitination assay RxxL) and the KEN box. indicated that RCS1 was ubiquitinated by both APC/C-Cdc20 The temporal control of APC/C activation, and hence the and APC/C-Cdh1 (Fig. 2A), similar to securin and cyclin B (data anaphase onset, is under complex regulation (1). Upon mitotic entry, APC/C is phosphorylated by mitotic kinases, such as Plk1, Author contributions: W.-m.Z., A.S., X.-l.C., J.R.Y., and G.F. designed research; W.-m.Z., which is necessary but not sufficient for its activation. On the other J.A.C., A.S., and X.-l.C. performed research; W.-m.Z., J.A.C., A.S., X.-l.C., and J.R.Y. contrib- hand, the spindle checkpoint, which delays the anaphase onset until uted new reagents/analytic tools; W.-m.Z., J.A.C., A.S., X.-l.C., J.R.Y., and G.F. analyzed data; all of the chromosomes are aligned at the metaphase plate, prevents and W.-m.Z., A.S., and G.F. wrote the paper. the activation of APC/C through association of inhibitory check- The authors declare no conflict of interest. point proteins Mad2 and BubR1 with APC/C-Cdc20 (6). This This article is a PNAS Direct Submission. checkpoint mechanism ensures the fidelity of chromosome segre- ‡To whom correspondence should be addressed. E-mail: [email protected]. gation and determines the timing of anaphase onset. This article contains supporting information online at www.pnas.org/cgi/content/full/ Given the central role of the APC/C in mitotic regulation, it is 0709227105/DCSupplemental. CELL BIOLOGY essential to identify its substrates to gain a mechanistic understand- © 2008 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0709227105 PNAS ͉ September 9, 2008 ͉ vol. 105 ͉ no. 36 ͉ 13415–13420 Downloaded by guest on October 1, 2021 Fig. 1. Substrates are specifically ubiquitinated by ECT2 RCS1 FLJ20345 APC/C-Cdh1 in vitro. iAPC/C was immunopurified from - Cdh1 + Cdh1 - Cdh1 + Cdh1 - Cdh1 + Cdh1 Xenopus interphase extracts and activated by in vitro translated Cdh1. APC/C-dependent ubiquitination of 35S-labeled substrates was analyzed in the presence of E1, E2, ubiquitin, ubiquitin-aldehyde, and ATP at indi- Ubiquitin cated times. The arrow points to the input substrates, Conjugates and the bracket indicates the position of ubiquitin conjugates. The extent of ubiquitination can be judged by the disappearance of the input substrates and by the formation of ubiquitin conjugates migrat- 0 15 30 60 0 15 30 60 0 15 30 60 0 15 30 60 0 15 30 60 0 15 30 60 (min) ing slower than the input substrates. not shown), whereas substrates degraded at the M-to-G1 tran- RCS1 protein stability during mitotic exit. The level of RCS1 was sition, such as Plk1, were efficiently ubiquitinated by APC/C- reduced before that of Cdh1 (Fig. 3A), which occurs in G1.To Cdh1, but weakly by APC/C-Cdc20 (Fig. 2A). determine the exact cell-cycle stage for RCS1 destruction, HeLa S3 RCS1 contains two putative D-boxes at amino acids 14–17 and cells were synchronized at prometaphase by a thymidine- 53–56, but no KEN box (Fig. 2B). The second D-box is conserved nocodazole block and then released into fresh media (Fig. 3B and among human, mouse, and Xenopus RCS1, whereas the first one is Fig. S2B). As expected, RCS1 levels were down-regulated during not. Mutation of the first D-box (DB1) (RxxL to AxxA) did not or mitotic exit, and the bulk of RCS1 was degraded after the destruc- only weakly affected the ubiquitination efficiency, whereas muta- tion of securin at anaphase but before the degradation of Plk1 in G1. tion of the second D-box (DB2) greatly reduced its ubiquitination To confirm that the APC/C-Cdh1 is required for the destruction by the APC/C-Cdh1 (Fig. 2C). Mutations of both D-boxes (DB1/2) of RCS1, we inhibited the activity of the APC/C-Cdh1 by RNAi.
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