Oncogene (1999) 18, 5131 ± 5137 ã 1999 Stockton Press All rights reserved 0950 ± 9232/99 $15.00 http://www.stockton-press.co.uk/onc Identi®cation of human APC10/Doc1 as a subunit of anaphase promoting complex

Yasuhiro Kurasawa1 and Kazuo Todokoro*,1

1Tsukuba Life Science Center, The Institute of Physical and Chemical Research (RIKEN), 3-1, Koyadai, Tsukuba, Ibaraki 305-0074, Japan

Anaphase-promoting complex or cyclosome (APC) is a (He et al., 1997; Li et al., 1997; Gorbsky et al., 1998; ubiquitin ligase which speci®cally targets mitotic reg- Kim et al., 1998; Hwang et al., 1998; Fang et al., ulatory factors such as Pds1/Cut2 and cyclin B. 1998b; Kallio et al., 1998), and regulation by Bub2 and Identi®cation of the subunits of multiprotein complex the RENT complex system (Visintin et al., 1998, 1999; APC in several species revealed the highly conserved Fesquet et al., 1999; Fraschini et al., 1999; Alexandru composition of APC from yeast to human. It has been et al., 1999; Shou et al., 1999). reported, however, that vertebrate APC is composed of Identi®cation of the subunits of multiprotein at least eight subunits, APC1 to APC8, while budding complex APC in several species revealed its highly yeast APC is constituted of at least 12 components, conserved composition from yeast to human (Peters et Apc1 to Apc13. It has not yet been clearly understood al., 1996; Zachariae et al., 1998a; Yu et al., 1998). whether additional components found in budding yeast, Vertebrate APC is composed of at least eight subunits, Apc9 to Apc13, are actually composed of mammalian APC1/Tsg24, APC2, APC3/CDC27Hs, APC4, APC5, APC. Here we isolated and characterized human APC6/CDC16Hs, APC7 and APC8/CDC23Hs (Peters APC10/Doc1, and found that APC10/Doc1 binds to et al., 1996; Yu et al., 1998). The APC of budding APC core subunits throughout the cell cycle. Further, it yeast is constituted of at least 12 components, Apc1, was found that APC10/Doc1 is localized in centrosomes Rsi1/Apc2, Cdc27/Apc3, Apc4, Apc5, Cdc16/Apc6, and mitotic spindles throughout mitosis, while it is also Cdc23/Apc8, Apc9, Doc1/Apc10, Apc11, Cdc26/ localized in kinetochores from prophase to anaphase and Apc12 and Apc13 (Zachariae et al., 1996, 1998a). It in midbody in telophase and cytokinesis. These results has not yet been clearly established whether or not strongly support the notion that human APC10/Doc1 additional components found in budding yeast, Apc9 may be one of the APC core subunits rather than the to Apc13, are actually composed of mammalian APC. transiently associated regulatory factor. APC10/DOC1, which was originally isolated by the enrichment of a mitotic arrest mutant of budding yeast, Keywords: APC; APC10; centromere; Doc1; ubiquitin was identi®ed as a gene encoding a component of cyclin proteolysis machinery (Irniger et al., 1995; Hwang and Murray, 1997). The doc1 mutants were arrested as large budded cells at 378C with a single Introduction nucleus and a short spindle, which were typical phenotypes in mitotic cyclin proteolysis mutants and Anaphase-promoting complex or cyclosome (APC) is a showed a defect in the ubiquitination of mitotic cyclin ubiquitin ligase which speci®cally targets the mitotic in G1-arrested cells (Hwang and Murray, 1997). regulatory factors (King et al., 1995; Sudakin et al., Indeed, identi®cation of budding yeast APC subunits 1995; Funabiki et al., 1996; Peters et al., 1996; revealed that Doc1 is one of the APC components Zachariae et al., 1998a; Yu et al., 1998). The APC (Zachariae et al., 1998a). Fission yeast apc10 mutant activity was found to be regulated by at least four was isolated as a G1 arrest mutant which showed distinct mechanisms; activation and inactivation by sterility and temperature-sensitive growth with a defect phosphorylation and dephosphorylation (Hershko et in chromosome segregation (Kominami et al., 1998). al., 1994; Lahav-Baratz et al., 1995; Sudakin et al., The apc10+ was essential for cell viability, and the 1995; Yamashita et al., 1996; Ishii et al., 1996; Peters et mutant showed two kinds of terminal morphology, al., 1996; Shirayama et al., 1998; Descombes and Nigg, elongated cells and cut phenotypes (Kominami et al., 1998; Charles et al., 1998; Kotani et al., 1998, 1999), 1998). It was found that Apc10 genetically and activation by the binding of substrate-speci®c WD- physically interacts with a universal subunit of the repeat proteins, Cdc20/p55CDC/Fizzy and Cdh1/Hct1/ APC, and is required for the ubiquitination of B type Fizzy-related (Visintin et al., 1997; Schwab et al., 1997; cyclin (Kominami et al., 1998). These results suggest Kallio et al., 1998; Fang et al., 1998a,b; Kramer et al., that Apc10/Doc1 is crucial for mitotic exit and for the 1998; Zachariae et al., 1998b; Lorca et al., 1998), degradation of mitotic cyclins; however, it was suppression of APC activity by the spindle assembly demonstrated that Apc10/Doc1 may not be a core checkpoint through the Mad family and Bub family component of APC. The majority but not all Doc1 population co-sedimented at 20S APC in a sucrose gradient in budding yeast (Hwang and Murray, 1997). In ®ssion yeast, myc-tagged Apc10 was not co- sedimented with APC, and the sedimentation pro®les *Correspondence: K Todokoro Received 26 June 1999; revised 9 August 1999; accepted 9 August of APC components appear normal in apc10 mutant 1999 (Kominami et al., 1998), whereas APC was physically Human APC10/Doc1 as a subunit of APC Y Kurasawa and K Todokoro 5132 disrupted in other mutations of APC components, such Expression of human APC10/Doc1 during cell cycle as cut4, cut9 and nuc2 (Yamashita et al., 1996; Yamada et al., 1997). In addition, APC10/Doc1 has not been A polyclonal rabbit antiserum was raised against identi®ed in vertebrate APC. human APC10/Doc1 fused to glutathione S-transfer- In spite of the rapid progress on the studies of ase, and puri®ed by anity chromatography of the regulation of APC activity, whole APC core compo- fusion protein. The puri®ed antibody detected APC10/ nents and their precise subcellular localization during Doc1 of 24 kDa in HeLa cell lysates in G1/S phase mitosis remains obscure. We therefore isolated and and M phase (Figure 2a). characterized human APC10/Doc1, and found that The expression level of APC10/Doc1 during the cell human APC10/Doc1 is one of the APC core cycle was examined by immunoblot analysis. HeLa components. cells were arrested at G1/S phase by aphidicolin, released, and then re-arrested by nocodazole at prometaphase. The cells were harvested at various time points after nocodazole release, and the cell Results extracts were probed with anti-APC10/Doc1 anti- body, anti-cyclin B1 antibody and anti-APC3/Cdc27 Isolation of human APC10/Doc1 cDNA antibody. The cyclin B1 was mostly degraded within A homology search of the human expressed sequence 60 min after nocodazole release (Figure 2b, upper tag (EST) data base identi®ed a cDNA fragment that is panel) as cells entered G1 phase. The supershift of signi®cantly homologous to APC10/Doc1 of budding APC3/Cdc27 by phosphorylation similarly diminished yeast and ®ssion yeast (Irniger et al., 1995; Hwang and within 60 min (Figure 2b, middle panel), while the level Murray, 1997; Kominami et al., 1998). Full-length of protein (sum of phosphorylated and unphosphory- human APC10/Doc1 cDNA (DDBJ/EMBL/GenBank lated forms) was unchanged. Figure 2b (lower panel) accession number AB012109) was isolated from a shows that the level of APC10/Doc1 was constant from cDNA library of human erythroleukemia K562 cells, prometaphase to G1/S phase, indicating that APC using an EST fragment ampli®ed by PCR as a probe. activity is not correlated with the protein level of Sequence analysis of seven independent clones revealed APC10/Doc1 during the cell cycle. that all cDNAs isolated encode the same open reading frame but have various amino-terminal sequences. APC10/Doc1 as a subunit of APC Thus the translational initiation site was con®rmed by cap site PCR. Figure 1 shows the con®rmed full- To examine whether or not mammalian APC10/Doc1 length human APC10/Doc1 sequence aligned with is one of the APC core subunits, the binding of those of budding yeast and ®ssion yeast. Human APC10/Doc1 with other APC components during the APC10/Doc1 cDNA encodes a peptide of 185 amino cell cycle was examined in HeLa cells by immunopre- acids with a predicted molecular mass of 21 252 cipitation followed by immunoblot analysis. The daltons and a predicted isoelectric point of 9.71. APC10/Doc1 was clearly detected in the immunopre- Human APC10/Doc1 shares 28.5% and 37.8% amino cipitates with anti-APC3/Cdc27 antibody in aphidico- acid sequence identity and 40.0% and 48.2% sequence lin-treated cells (G1/S phase) (Figure 3a, lane 2), similarity with those of budding yeast and ®ssion yeast, nocodazole-treated cells (prometaphase) (Figure 3a, respectively. lane 3), and cells within 45 min after nocodazole

Figure 1 Sequence alignments of human APC10/Doc1 with corresponding yeast proteins. Identical amino acid residues are shown on a black background. Conservative amino acid substitutions are shown on a gray background Human APC10/Doc1 as a subunit of APC Y Kurasawa and K Todokoro 5133

Figure 2 Expression level of human APC10/Doc1 during cell cycle. (a) Cell lysates of aphidicolin-treated (G1/S phase) (lane 1) or nocodazole-treated (prometaphase) HeLa cells were immunoblotted with anti-human APC10/Doc1 antibody. Arrow indicates APC10/Doc1. (b) HeLa cells were arrested at G1/S phase by aphidicolin, released, and then re-arrested by nocodazole at prometaphase. The cells were harvested at the indicated time points after nocodazole release, and equal amounts of the cell extracts were probed with anti-cyclin B1 antibody (upper panel), anti-APC3/Cdc27 antibody (middle panel) and anti-APC10/Doc1 antibody (lower panel)

release (late mitosis) (Figure 3a, lane 4), but not in the dots on the chromosomes were kinetochores of the immunoprecipitate with non-immune serum (Figure 3a, sister chromatids (Figure 5, lane 1). In triple stainings lane 1). Conversely, both APC3/Cdc27 and APC6/ (APC10/Doc1, centromere and DNA), the double dots Cdc16 were detected in the immunoprecipitates with in a centromere staining could be clearly seen (Figure anti-APC10/Doc1 antibody in the same cell extracts 5, lane 1, bottom panel). In metaphase, APC10/Doc1 (data not shown, see below). was localized in centrosomes and in kinetochores, while Furthermore, the cell extracts were prepared at the kinetochore stainings became faint (Figure 4, lane various time points after nocodazole release, and the 2). In anaphase A, APC10/Doc1 was localized in the immunoprecipitates with anti-APC10/Doc1 antibody spindle midzone, centrosomes and spindles (Figure 4, were probed with anti-APC3/Cdc27 antibody (Figure lane 3). In anaphase B, it was primarily in the spindle 3b, upper panel) or anti-APC6/Cdc16 antibody (Figure midzone, and the stainings of the spindles and spindle 3b, lower panel). The amounts of immunoprecipitated poles became much weaker (Figure 4, lane 4). The APC10/Doc1 at each time point were constant (data kinetochores were only weakly stained during anaphase not shown), and no APC subunit was detected by non- (Figure 5, lane 2). In telophase and cytokinesis, immune serum (Figure 3b, lane 1). It was found that APC10/Doc1 was mainly localized in midbody both APC3/Cdc27 and APC6/Cdc16 were always (Figure 4, lane 5). In addition to the speci®c associated with APC10/Doc1 through prometaphase localization to centrosomes, kinetochores and spin- to S phase (Figure 3b). These results indicate that dles, APC10/Doc1 was di€usely distributed in the APC10/Doc1 may be one of the APC core components cytoplasm throughout the mitosis (Figure 4), while it rather than a transiently associated regulatory factor. was detected nowhere in interphase (data not shown).

Subcellular localization of APC10/Doc1 Discussion It has been described that two human APC components, APC3/Cdc27 and APC6/Cdc16 are co- It was reported that the majority, but not all (or any) localized in centrosomes at all stages of the cell cycle of the APC10/Doc1 population co-sedimented at 20S and in mitotic spindles during mitosis (Tugendreich et APC in a sucrose gradient in budding (or ®ssion) yeast al., 1995), whereas mouse APC1/Tsg24 is localized in (Hwang and Murray, 1997; Kominami et al., 1998). centromeres (Jorgensen et al., 1998). To determine the Further, Xenopus APC10 homologue was not detected exact subcellular localization of APC10/Doc1 during in APC which was puri®ed by immunoprecipitation the cell cycle, we performed indirect immunofluores- with anti-APC3/Cdc27 antibody (Peters et al., 1996). cence microscopic analyses in HeLa cells during These results suggested that Doc1/Apc10 might be a mitosis. In early prophase, APC10/Doc1 was localized factor which transiently associates with APC in a cell in the duplicated centrosomes and mitotic spindles cycle- or substrate-speci®c manner. In this study, (data not shown). In prometaphase, besides two however, we described that human APC10/Doc1 spindle poles and mitotic spindles, a number of paired always binds to APC and its protein level was APC10/Doc1 stainings on the chromosomes were constant throughout the cell cycle. Further, we found clearly visible (Figure 4, lane 1). Double stainings of that the APC immunopuri®ed with anti-APC10/Doc1 the cells with anti-centromere antibody together with antibody included stoichiometric amounts of APC10/ anti-APC10/Doc1 antibody revealed that these paired Doc1 with APC core components in a silver stained gel Human APC10/Doc1 as a subunit of APC Y Kurasawa and K Todokoro 5134

Figure 3 Human APC10/Doc1 as an APC subunit. (a) Human APC was immunoprecipitated with anti-APC3/Cdc27 antibody from the cell extracts of aphidicolin-treated (lane 2), nocodazole-treated (lane 3) or nocodazole-treated and released (45 min after release) (lanes 1 and 4) HeLa cells, and was probed with anti-APC10/Doc1 antibody. Rabbit non-immune serum was used as a control precipitation (lane 1). Arrow indicates APC10/Doc1. (b) HeLa cells were released from nocodazole block, and the cell extracts prepared at the indicated time points were immunoprecipitated with anti-APC10/Doc1 antibody (lanes 2 ± 9) or with non-immune serum (lane 1). The immunoprecipitates were probed with anti-APC3/Cdc27 antibody (upper panel) or anti-APC6/Cdc16 antibody (lower panel)

(data not shown). In the case of Xenopus APC10/Doc1, the centrosomes throughout the cell cycle, and to the it might not have been detected in SDS ± PAGE mitotic spindles during mitosis (Tugendreich et al., because of its small size. Therefore, we concluded 1995). In contrast, APC1/Tsg24 was detected in that human APC10/Doc1 may be a core component of centromeres from prophase to anaphase in Chinese APC rather than a transiently associated regulatory hamster ovary CHO cells, while it was constitutively factor. Highly conserved sequences of APC10/Doc1 of associated with centromeres in mouse Swiss-3T3 various species (from yeast to human) also support this ®broblasts (Jorgensen et al., 1998). We also con®rmed notion, while we cannot completely exclude the the subcellular localization of these APC subunits by possibility that APC10/Doc1 is a substoichiometric the same antibodies (data not shown). Taken together, component of APC. these results suggest the following notion. The APC on We have shown in this paper that the subcellular the motor proteins may move along mitotic spindles localization of human APC10/Doc1 dynamically between centrosomes and centromeres, and locate at changes during mitosis. APC10/Doc1 was localized in the sites where the target molecules and/or regulatory centrosomes and mitotic spindles throughout mitosis, factors of APC exist at a certain time point. The APC while it was in kinetochores from prophase to in kinetochores may ubiquitinate Cut2/Pds1 at anaphase, in midzone in anaphase, and midbody in metaphase/anaphase transition, and one in midbody telophase and cytokinesis. It has been reported that may ubiquitinate Cyclin B, Cdc20, Ase1 and Plk which human CDC27Hs and CDC16Hs are co-localized to are known to be degraded during anaphase, telophase Human APC10/Doc1 as a subunit of APC Y Kurasawa and K Todokoro 5135

Figure 4 Subcellular localization of human APC10/Doc1 during mitosis. HeLa cells in prometaphase (lane 1), metaphase (lane 2), anaphase A (lane 3), anaphase B (lane 4) or telophase (lane 5) were stained with anti-APC10/Doc1 rabbit antiserum (top panels), anti- tubulin rat antibody (the second panels), and DAPI (the third panels). The Cy3-conjugated anti-rabbit or ¯uorescein-conjugated anti-rat IgGs were used as a secondary antibody. The merges of all three ¯uorochromes are also shown (Triple) (bottom panels)

and cytokinesis (Koepp et al., 1999; Morgan, 1999; block cells at the G1/S boundary. To arrest cells in mitosis, Weinstein, 1997; Descombes and Nigg, 1998; Jiang et they were treated with 1 mg/ml aphidicolin for 20 h, washed al., 1998). Indeed, some of these substrates (or with PBS twice, incubated with fresh medium for 5 h and regulatory factors) have been described as being co- then blocked with medium containing 12 ng/ml nocodazole localized with APC at the right time point (Golsteyn et (Janssen) for 8 h. Mitotic cells were mechanically shaken o€ from the culture ¯asks and collected. For the nocodazole- al., 1995; Weinstein, 1997; Jiang et al., 1998). block and release experiment, the mitotic shaken-o€ cells The role of APC10/Doc1 in APC activity is not were washed twice with PBS prewarmed at 378C, incubated known, but we also found that the APC immunopre- in prewarmed fresh medium for 20 min to 10 h, and then cipitated with anti-APC10/Doc1 antibody contained harvested. whole APC core components, but it could not function as the ubiquitination machinery of cyclin B due to the cDNA cloning and sequencing inhibition of APC activity by the antibody (data not shown). These results are also consistent with the A partial human cDNA fragment encoding an open reading previous reports that APC10/Doc1 is essential compo- frame with homology to budding yeast Doc1 (ScDOC1) was nent in APC activity (Irniger et al., 1995; Hwang and identi®ed from a search of the NCBI dbEST using the BLAST algorithm. An EST clone (clone 668857) that Murray, 1997; Kominami et al., 1998). Taken together, signi®cantly matched ScDOC1 was sequenced. The radi- we concluded that APC10/Doc1 is one of the core olabeled cDNA fragment was used for screening a lgt10 subunits of APC which may be necessary for its cDNA library of human erythroleukemia K562 cells activity. (Clontech). The obtained full-length human APC10/Doc1 cDNAs were then sequenced. The 5' end sequence was con®rmed by the sequencing of the Cap Site cDNA (Nippon Gene). Materials and methods

Cell culture and synchronization Immunoprecipitation and immunoblotting HeLa cells were grown in Dulbecco's modi®ed Eagle's The GST-fused full-length APC10/Doc1 in pGEX-2T was medium (Gibco±BRL) supplemented with 10% fetal bovine expressed in XL1-Blue MRF' with 0.4 mM isopropyl b-D- serum. For synchronization with a double aphidicolin block, thiogalactopyranoside and isolated by anity chromatogra- exponentially growing cells were arrested in the presence of phy on glutathione-Sepharose (Pharmacia). The puri®ed GST 1 mg/ml aphidicolin (Wako Chemicals) for 18 h, washed with fusion protein was used to immunize a rabbit. The anti- phosphate-bu€ered saline (PBS), and incubated in fresh APC10/Doc1 rabbit serum was anity-puri®ed with GST- medium without aphidicolin for 8 h. Cells were then APC10/Doc1 fusion protein coupled to HiTrap NHS- incubated with 1 mg/ml aphidicolin for additional 18 h to activated column (Pharmacia). Human APC10/Doc1 as a subunit of APC Y Kurasawa and K Todokoro 5136 with TBS-T (TBS with 0.5% Tween-20), and incubated either with anity puri®ed rabbit anti-APC10/Doc1 antibody (4 mg/ ml), rabbit anti-APC3/Cdc27 antiserum (1 : 6000 dilution), rabbit anti-APC6/Cdc16 antiserum (Tugendreich et al., 1995) (1 : 800 dilution), or mouse anti-human cyclin B1 antibody (Upstate Biotechnology) (1 : 2000 dilution) in TBS-T for 2 h at room temperature. The membrane was then washed four times with TBS-T, incubated with HRP-conjugated anti- rabbit and anti-mouse antibodies (Jackson ImmunoResearch Laboratories) for 1 h in TBS-T, washed three times with TBS-T, and then developed by enhanced chemiluminescence detection (Amersham).

Immunostaining HeLa cells on coverslips were rinsed twice at room temperature with PHEM (60 mM PIPES, 25 mM HEPES,

pH 6.9 with KOH, 10 mM EGTA, 2 mM MgCl2) and lysed with 0.5% Triton X-100 in PHEM for 1 min at room temperature. The cells on coverslips were ®xed with cold (7208C) methanol for 5 min, immediately dried, and washed brie¯y with PBS. The coverslips were incubated in primary antibodies in an antibody solution (0.1 M PIPES ± KOH,

pH 7.2, 1 mM MgSO4,1mM EGTA, 1.83% L-lysine, 1%

BSA, 0.1% NaN3) for 45 min at 378C. Primary antibodies used were: rabbit anti-APC10/Doc1 antiserum (1 : 200 dilution), human anti-centromere autoimmune serum (1 : 100 dilution) (Nagata et al., 1997), or rat anti-tubulin antibody (Biosys) (1 : 20 dilution). After the wash with PBS, the coverslips were incubated with Cy3-conjugated anti-rabbit antibody (Jackson ImmunoResearch Laboratories) (1 : 250 dilution), and ¯uorescein isothiocyanate-conjugated anti-rat antibody (Jackson ImmunoResearch Laboratories) (1 : 50 dilution) or anti-human antibody (Jackson ImmunoResearch Laboratories) (1 : 50 dilution) in antibody solution containing Figure 5 APC10/Doc1 in kinetochores in prometaphase. HeLa 1 mg/ml 4,6-diamidino-2-phenylindole (DAPI) for 45 min at cells in prometaphase (left panels) and anaphase (right panels) 378C, washed with PBS, and mounted on slides with 90% were stained with rabbit anti-APC10/Doc1 antiserum (top panels) glycerol, and 10% 106PBS containing para-phenylenedia- or human anti-centromere autoimmune serum (the second mine. Samples were observed using an Olympus ¯uorescence panels). The Cy3-conjugated anti-rabbit or ¯uorescein-conju- microscope (BX60) and photographed. gated anti-human IgG was used as secondary antibody

Cells were washed with PBS twice and lysed with a lysis Note added in proof bu€er (50 mM Tris-HCl, pH 7.5, 0.5% NP-40, 150 mM NaCl, The accession number of human APC10/Doc1 is 2mM sodium orthovanadate, 100 mM sodium ¯uoride, 1 mM ABO12109. Grossberger et al. (1999) also reported the sodium pyrophosphate, 4 mM EDTA) including protease characterization of APC10/Doc1. inhibitors (40 mM Pefablock, 1 mg/ml pepstatin A, 10 mg/ml leupeptin, 1 mg/ml aprotinin) and 100 nM microcystine LR at 48C for 30 min. The lysates were precleared with Protein A- Acknowledgments Sepharose beads and used for immunoprecipitations. Anti- We thank P Hieter for anti-Cdc27 and anti-Cdc16 bodies were covalently coupled to Protein A beads as antibodies, and Y Muro for anti-centoromere antibody. described (Harlow and Lane, 1998). Ten ml of anti-APC10/ We also thank T Toda and T Oyamatsu for valuable Doc1 antibody or anti-APC3/Cdc27 antibody (Tugendreich discussion, and M Fujii and M Nishida for technical et al., 1995) beads were incubated with 250 mg of HeLa cell assistance. This work was supported in part by a Special lysates at 48C for 3 h. The proteins were resolved by SDS ± Grant for Promotion of Research from the Institute of PAGE, and transferred to nitrocellulose membrane. The Physical and Chemical Research (RIKEN) and grants from membrane was blocked in 5% BSA or 5% skim milk in TBS the Ministry of Education, Science and Culture of Japan (20 mM Tris-HCl, pH 7.6, 137 mM NaCl) for 8 h, washed and from the Science Technology Agency of Japan.

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