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Cdc20 Associates with the Kinase Aurora2/Aik

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Cdc20 Associates with the Kinase Aurora2/Aik Proc. Natl. Acad. Sci. USA Vol. 96, pp. 7306–7311, June 1999 Cell Biology Cdc20 associates with the kinase aurora2yAik DAWN C. FARRUGGIO,FIONA M. TOWNSLEY*, AND JOAN V. RUDERMAN† Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115 Contributed by Joan V. Ruderman, April 22, 1999 ABSTRACT Cdc20yfizzy family proteins are involved in cannot be activated. It appears that once Mad2 is released, activation of the anaphase-promoting complexycyclosome, Cdc20 permits APCyC activation (22, 23). which catalyzes the ubiquitin-dependent proteolysis of cell A second Cdc20-related set of proteins includes Cdh1yHct1 cycle regulatory proteins such as anaphase inhibitors and in S. cerevisiae, Fzr in Drosophila, X-Fzr in Xenopus, and mitotic cyclins, leading to chromosome segregation and exit Hs-FzryhCdh1 in human (refs. 10, 19, 32, and 37; I. Dawson, from mitosis. Previous work has shown that human Cdc20 personal communication). In animal cells, this second set of (hCdc20yp55CDC) associates with one or more kinases. We proteins, collectively termed fizzy-related (Fzr), is proposed to y report here that Cdc20-associated myelin basic protein kinase maintain the active state of the APC CinG1, after Cdc20 activity peaks sharply in early M phase (embryonic cells) or destruction (19, 20). In yeast, both Cdc20 and Cdh1 act during in G2 phase (somatic cells). In HeLa cells, Cdc20 is associated mitosis, but at different times, and they have been proposed to with the kinase aurora2yAik. Aurora2yAik is a member of the function as specificity factors for different APCyC substrates y aurora Ipl1 family of kinases that, like Cdc20, previously has (25, 32, 38). been shown to be localized at mitotic spindle poles and is In mammalian cells, Cdc20 proteins have one or more involved in regulating chromosome segregation and maintain- associated kinase activities (30). Here, we report that human ing genomic stability. The demonstration that Cdc20 is asso- Cdc20 associates with aurora2yAik, a member of the auroray y ciated with aurora2 Aik suggests that some function of Cdc20 Ipl1 family of kinases that, like Cdc20, localizes to the mitotic is carried out or regulated through its association with spindle poles and has been implicated previously in pathways y aurora2 Aik. regulating chromosome segregation (39–47). We find that y Cdc20-associated aurora2 Aik activity peaks during G2 phase Accurate segregation of sister chromatids during mitosis is in somatic cells and is significantly lower in both unperturbed critical for production of two viable, nontumorigenic daughter mitotic cells collected by mitotic shake-off and in nocodazole- cells. Mitosis is initiated by the mitotic cyclinycdk complexes arrested M-phase cells, in which the APCyC activity is sup- and ends with the ubiquitin-dependent destruction of the pressed by checkpoint control mechanisms. It is possible that y cyclin subunits and other inhibitors of the M G1 transition. Cdc20 could act as a targeting subunit for aurora2yAik. This destruction pathway consists of a ubiquitin-activating Alternatively, aurora2yAik could function by phosphorylating enzyme (E1), a ubiquitin-conjugating enzyme (E2), a ubiquitin Cdc20, thereby influencing the activity of Cdc20. These pos- ligase (E3), and the 26S proteasome that degrades ubiquiti- sibilities now can be tested in a variety of experimental systems. nated proteins (1, 2). For cyclins and certain other mitotic targets, the E3 activity is provided by the anaphase promoting complexycyclosome (APCyC) and is regulated during the cell EXPERIMENTAL PROCEDURES cycle (refs. 3–5 and reviewed in refs. 6–8). Activation of the Isolation of Clam Cdc20 cDNAs. cDNAs were isolated from y y APC C is initiated, after a lag phase, by cyclin B cdk1 and a clam oocyte cDNA library in lZAPII, made from unstimu- y appears to involve additional kinases (9, 10). APC C activa- lated, full-grown clam oocytes by using the ZAP-cDNA Syn- y tion has been correlated with phosphorylation of APC C thesis Kit (Stratagene). A fragment of clam Cdc20 (FT11) was subunits Cdc16, Cdc27, and Tsg24 by Plk1 (10–15). In addi- 9 y y obtained by thermocycling using primer C20F3 [5 -TIGCI(T tion, the APC C receives inhibitory phosphorylations through A)(CyG)IGGIGGIAA(CyT)GA(CyT)AA-39] corresponding protein kinase A (11, 16, 17). y to the LASGGNDN sequence conserved in human, Drosoph- Cdc20 Fizzy (Fzy) family proteins also are required for ila, and S. cerevisiae Cdc20, along with the vector primer, T7, activation of the APCyC (10, 18–25). Mutants of Cdc20 were 9 2 found at the 3 end of all the cDNAs. A subclone of FT11 described first in Saccharomyces cerevisiae, where cdc20 cells (FT11B3) containing the 39 end of the ORF was used to screen are defective in several microtubule-mediated processes (26– 1 phage plaques for full-length cDNAs. Three cDNAs yielded 28). Cdc20 homologs in other organisms include slp1 in identical coding sequences except foraCtoAchange at fission yeast, Fzy in Drosophila, X-Fzy in Xenopus, and nucleotide 466, which does not change the amino acid se- y Drosophila p55CDC hCdc20 in humans (18, 23, 29, 30). In ,Fzy quence. mutants fail to separate sister chromatids or degrade mitotic Antibodies. His6-tagged clam or human (30) Cdc20 proteins cyclins, a finding that led to the hypothesis that Cdc20 is 1 were purified from bacteria by using Ni2 -NTA agarose (Qia- required for activation of the APCyC (18, 31). Cdc20 has been gen, Chatsworth, CA). The recombinant His -tagged Cdc20 shown to play a critical, but not fully understood, role in mitotic 6 was solubilized in solutions containing 8 M urea, further activation of the APCyC (10, 20, 22, 24, 25, 32). Cdc20 also has been implicated in the DNA damage and spindle assembly Abbreviations: MBP, myelin basic protein; APCyC, anaphase- checkpoints. Cdc20 binds Mad2 along with Mad1 and Mad3, y components of the spindle assembly checkpoint pathway (21, promoting complex cyclosome; GVBD, germinal vesicle breakdown. y Data deposition: The Spisula solidissima (surf clam) Cdc20 nucleotide 29, 33–36). When Cdc20 is complexed with Mad2, the APC C sequence reported in this paper has been deposited in the GenBank database (accession no. AF052575). The publication costs of this article were defrayed in part by page charge *Present address: Division of Cell Biology, Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH, payment. This article must therefore be hereby marked ‘‘advertisement’’ in United Kingdom. accordance with 18 U.S.C. §1734 solely to indicate this fact. †To whom correspondence should be addressed. e-mail: PNAS is available online at www.pnas.org. [email protected]. 7306 Downloaded by guest on September 25, 2021 Cell Biology: Farruggio et al. Proc. Natl. Acad. Sci. USA 96 (1999) 7307 purified by SDSyPAGE (48), and used to generate rabbit Sucrose solutions were made in 50 mM Hepes NaOH, pH 7.3, polyclonal antibodies. 1 mM DTT for clam oocyte samples or 50 mM Hepes KOH, Polyclonal antipeptide antibodies recognizing human auro- pH 7.3, 100 mM KCl, 1 mM MgCl2, 1 mM DTT for HeLa ra2yAik were the gift of G. Gopalan and P. Donovan (Thomas extracts. Five-milliliter gradients were centrifuged 18 hr at Jefferson University, Philadelphia) (ref. 41 and unpublished 27,000 rpm (68,000 3 g) and 4°C in a Sorvall AH650 rotor (4). work). Antibodies to human p55CDC and human cyclins were Twenty-four fractions of 200–220 ml were collected. purchased from Santa Cruz Biotechnology. Rabbit polyclonal antibodies to human Cdc16 and Cdc27 were a gift of P. Hieter RESULTS (Johns Hopkins School of Medicine, Baltimore) (49). Immune blotting was done as in ref. 50. Cdc20 Protein Levels Do Not Vary During the Meiotic or Oocyte Extracts. Clam oocytes were collected and washed Early Mitotic Cycles of Clam Oocytes. Because the naturally according to refs. 51 and 52. Oocytes (48,000 oocytesyml) were synchronous cell cycles of early embryos provide good oppor- fertilized, and 1-ml aliquots were collected at 5-min intervals tunities for examining cell cycle stage-specific activities of for protein gel samples; 100-ml aliquots were orcein stained proteins uncomplicated by the effects of synchronizing re- and examined microscopically to determine cell cycle position agents, we wanted to examine Cdc20 and the Cdc20-associated (52). For extracts, '3 ml of packed oocytes (3 3 107 cells) in kinase activity across the meiotic and mitotic cell cycles of the 200 ml of sea water was activated by fertilization. Extracts were clam Spisula solidissima. Three independently isolated clam prepared as described (51) from G2-arrested oocytes (unfer- Cdc20 cDNA clones were found to encode the same 522-aa tilized), GVBD meiosis I (germinal vesicle breakdown visible, protein (GenBank accession no. AF052575). Clam Cdc20 is early meiosis I), interphase 1 (both polar bodies and pronuclei very similar to human (48%), Xenopus (49%), and Drosophila with decondensed chromosomes visible), early mitosis 1 (con- (42%) Cdc20 proteins and less similar to Schizosaccharomyces densed chromosomes, occasional metaphase plate), late mi- pombe slp11 (36%) and S. cerevisiae Cdc20 (25%) proteins tosis 1 (anaphase underway, 5-min postmetaphase), mitosis 1 (see Fig. 6, which is published as supplemental data on the arrest (colchicine-arrested), interphase 2 arrest (emetine- PNAS web site, www.pnas.org). The conservation is particu- arrested at the two-cell stage with decondensed chromo- larly strong in the C-terminal region of the protein, which somes). consists of seven WD-40 domains. Clam Cdc20 is less similar HeLa Cell Extracts. HeLa cells were cultured, and whole- to Xenopus and Drosophila fizzy-related (35%) and human cell lysates were prepared as described (53). For synchroniza- Cdh1 (34%), indicating that it is a member of the Cdc20yfizzy tion, cells were treated with 2 mM thymidine for 18–24 hr, group.
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