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• W2078702698 abstract "We reported here an efficient and generally applicable genomic analysis that uses transcriptional profiling to identify candidate substrates of regulatory enzymes, such as kinases and ubiquitin ligases. We applied this strategy to the anaphase-promoting complex/cyclosome (APC/C), a ubiquitin ligase that controls sister chromatid separation and exit from mitosis. We found that a microtubule-associated protein, CKAP2, is a substrate of APC/C and demonstrated that ubiquitination and degradation of CKAP2 in vitro require a KEN-box and is mediated by Cdh1, an activator of APC/C. We showed that the levels of CKAP2 fluctuated across the cell cycle in culture cells, high in mitosis and low during mitotic exit. Overexpression of Cdh1 reduced the levels of CKAP2 in a KEN-box-dependent manner, while knockdown of Cdh1 increased the half-life of CKAP2. CKAP2 associated with centrosomal microtubules in late G2, but only after the separation of the duplicated centrosomes. During mitosis, CKAP2 associated with spindle poles and with spindle microtubules from prophase through anaphase and dis-appeared from microtubules during cytokinesis. The function of CKAP2 during mitosis does not seem essential, as efficient knockdown of CKAP2 neither altered the cell cycle distribution of the cells, nor generated observable mitotic defects. On the other hand, ectopic expression of either the wild-type or a non-degradable CKAP2 led to a mitotic arrest with monopolar spindles containing highly bundled microtubules. We concluded that CKAP2 is a physiological substrate of APC/C during mitotic exit and that a tight regulation of the CKAP2 protein level is critical for the normal mitotic progression. We reported here an efficient and generally applicable genomic analysis that uses transcriptional profiling to identify candidate substrates of regulatory enzymes, such as kinases and ubiquitin ligases. We applied this strategy to the anaphase-promoting complex/cyclosome (APC/C), a ubiquitin ligase that controls sister chromatid separation and exit from mitosis. We found that a microtubule-associated protein, CKAP2, is a substrate of APC/C and demonstrated that ubiquitination and degradation of CKAP2 in vitro require a KEN-box and is mediated by Cdh1, an activator of APC/C. We showed that the levels of CKAP2 fluctuated across the cell cycle in culture cells, high in mitosis and low during mitotic exit. Overexpression of Cdh1 reduced the levels of CKAP2 in a KEN-box-dependent manner, while knockdown of Cdh1 increased the half-life of CKAP2. CKAP2 associated with centrosomal microtubules in late G2, but only after the separation of the duplicated centrosomes. During mitosis, CKAP2 associated with spindle poles and with spindle microtubules from prophase through anaphase and dis-appeared from microtubules during cytokinesis. The function of CKAP2 during mitosis does not seem essential, as efficient knockdown of CKAP2 neither altered the cell cycle distribution of the cells, nor generated observable mitotic defects. On the other hand, ectopic expression of either the wild-type or a non-degradable CKAP2 led to a mitotic arrest with monopolar spindles containing highly bundled microtubules. We concluded that CKAP2 is a physiological substrate of APC/C during mitotic exit and that a tight regulation of the CKAP2 protein level is critical for the normal mitotic progression. Ubiquitin-mediated proteolysis plays key regulatory functions in diverse biological processes, ranging from cell cycle control, cell signaling, transcriptional regulation, immune-response to development (1Ciechanover A. Angew. Chem. Int. Ed. Engl. 2005; 44: 5944-5967Crossref PubMed Scopus (122) Google Scholar, 2Hershko A. Ciechanover A. Varshavsky A. Nat. Med. 2000; 6: 1073-1081Crossref PubMed Scopus (572) Google Scholar). The anaphase-promoting complex/cyclosome (APC/C) 2The abbreviations used are: APC/C, anaphase-promoting complex/cyclosome; FACS, fluorescence-activated cell sorter; siRNA, small interfering RNA; shRNA, small hairpin RNA; PBS, phosphate-buffered saline; GFP, green fluorescent protein; D-box, destruction box; MAPK, mitogen-activated protein kinase. is a key ubiquitin ligase that controls several transitions in the cell cycle (3Harper J.W. Burton J.L. Solomon M.J. Genes Dev. 2002; 16: 2179-2206Crossref PubMed Scopus (424) Google Scholar, 4Peters J.M. Mol. Cell. 2002; 9: 931-943Abstract Full Text Full Text PDF PubMed Scopus (778) Google Scholar, 5Peters J.M. Nat. Rev. Mol. Cell. Biol. 2006; 7: 644-656Crossref PubMed Scopus (1027) Google Scholar). APC/C-dependent degradation of cyclin A allows cells to progress from prophase to metaphase and the degradation of securin by the APC/C triggers the chromosome separation and anaphase onset. Late in anaphase, the destruction of cyclin B1 leads to the inactivation of the Cdk1 kinase activity and exit from mitosis. The APC/C also recognizes and degrades anillin, TPX2, Aurora A, Aurora B, and Plk1 during cytokinesis, thereby allowing an ordered transition into G1 (6Stewart S. Fang G. Cancer Res. 2005; 65: 8730-8735Crossref PubMed Scopus (125) Google Scholar, 7Stewart S. Fang G. Mol. Cell. Biol. 2005; 25: 10516-10527Crossref PubMed Scopus (82) Google Scholar, 8Zhao W.M. Fang G. J. Biol. Chem. 2005; 280: 33516-33524Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). In addition, the APC/C path-way has been linked to the control of DNA replication, and components of the prereplication complexes, such as Cdc6 and Cdt1, and an inhibitor of DNA replication, geminin, have all been shown as substrates of the APC/C (9Petersen B.O. Wagener C. Marinoni F. Kramer E.R. Melixetian M. Denchi E.L. Gieffers C. Matteucci C. Peters J.M. Helin K. Genes Dev. 2000; 14: 2330-2343Crossref PubMed Scopus (233) Google Scholar, 10Li A. Blow J.J. EMBO J. 2005; 24: 395-404Crossref PubMed Scopus (115) Google Scholar, 11McGarry T.J. Kirschner M.W. Cell. 1998; 93: 1043-1053Abstract Full Text Full Text PDF PubMed Scopus (741) Google Scholar). Thus, APC/C-mediated proteolysis couples the S phase with the completion of mitosis and contributes to mechanisms ensuring that the genome is replicated only once per cell cycle. The activity of the APC/C is tightly regulated in the cell cycle: The APC/C becomes active from prometaphase until the end of G1. One of the main regulatory mechanisms for the APC/C is through its association with accessory-activating factors, Cdc20/fizzy and Cdh1/fizzy-related. Both Cdc20 and Cdh1 directly bind to and activate the APC/C ligase. Cdc20 associates with the APC/C from prometaphase to anaphase, responsible for the degradation of cyclin A, cyclin B, and securin, whereas Cdh1 maintains the activity of the APC/C from late anaphase through G1, targeting multiple substrates for degradation (12Kramer E.R. Gieffers C. Holzl G. Hengstschlager M. Peters J.M. Curr. Biol. 1998; 8: 1207-1210Abstract Full Text Full Text PDF PubMed Google Scholar, 13Fang G. Yu H. Kirschner M.W. Mol Cell. 1998; 2: 163-171Abstract Full Text Full Text PDF PubMed Scopus (426) Google Scholar). In addition, phosphorylation of the APC/C by mitotic kinases, such as Plk1, also contributes to its mitotic activation (5Peters J.M. Nat. Rev. Mol. Cell. Biol. 2006; 7: 644-656Crossref PubMed Scopus (1027) Google Scholar). On the other hand, negative regulation of the APC/C by inhibitory proteins, such as Emi1, BubR1, and Mad2, determines the precise temporal activity of the APC/C during mitosis (14Fang G. Yu H. Kirschner M.W. Genes Dev. 1998; 12: 1871-1883Crossref PubMed Scopus (496) Google Scholar, 15Fang G. Mol. Biol. Cell. 2002; 13: 755-766Crossref PubMed Scopus (260) Google Scholar, 16Tang Z. Bharadwaj R. Li B. Yu H. Dev Cell. 2001; 1: 227-237Abstract Full Text Full Text PDF PubMed Scopus (346) Google Scholar, 17Sudakin V. Chan G.K. Yen T.J. J. Cell Biol. 2001; 154: 925-936Crossref PubMed Scopus (673) Google Scholar, 18Reimann J.D. Freed E. Hsu J.Y. Kramer E.R. Peters J.M. Jackson P.K. Cell. 2001; 105: 645-655Abstract Full Text Full Text PDF PubMed Scopus (310) Google Scholar). The APC/C recognizes two motifs in its substrates: the destruction box (D-box; RXXL where X is any amino acid) and the KEN-box (19Pfleger C.M. Kirschner M.W. Genes Dev. 2000; 14: 655-665Crossref PubMed Google Scholar, 20Glotzer M. Murray A.W. Kirschner M.W. Nature. 1991; 349: 132-138Crossref PubMed Scopus (1903) Google Scholar). Although the exact biochemical mechanism for substrate recognition remains to be characterized, it has been reported that APC/C-Cdc20 recognizes D-box-containing substrates and that APC/C-Cdh1 recognizes both D-box and KEN-box-containing substrates (19Pfleger C.M. Kirschner M.W. Genes Dev. 2000; 14: 655-665Crossref PubMed Google Scholar). A key question in understanding the function of the APC/C is to identify its substrates and to investigate their physiological function and the biological importance of their degradation. To develop a general approach for identification of APC/C substrates, we analyzed the transcriptional profiles of the known APC/C substrates involved in the cell cycle regulation and found that these substrates had expression profiles that co-varied with those of the known cell cycle regulators in tumor tissues, indicating that these substrates are indeed components of the cell cycle machinery whose expression is under selective pressures for co-variation during tumorigenesis. In addition, for the substrates that function in mitosis and cytokinesis, we found that the majority of them were transcriptionally induced in G2 or in mitosis. Thus, we searched for new candidate mitotic APC/C substrates whose expression co-varied with those of known cell cycle regulators in tumor tissues and whose expression was induced in G2/M in the cell cycle. Physiological substrates were then identified from this list of candidate mitotic regulators using an in vitro ubiquitination assay. In principle, this strategy is generally applicable to the identification of substrates for regulatory enzymes, such as ubiquitin ligases and kinases. Using this method, we have identified 9 new substrates of the APC/C and reported here the characterization of one of the substrates, CKAP2. CKAP2 was initially reported as a cytoskeleton-associated protein that is up-regulated in human gastric adenocarcinomas as well as in various tumor-derived cell lines (21Maouche-Chretien L. Deleu N. Badoual C. Fraissignes P. Berger R. Gaulard P. Romeo P.H. Leroy-Viard K. Oncogene. 1998; 17: 1245-1251Crossref PubMed Scopus (36) Google Scholar, 22Bae C.D. Sung Y.S. Jeon S.M. Suh Y. Yang H.K. Kim Y.I. Park K.H. Choi J. Ahn G. Park J. J. Cancer Res. Clin. Oncol. 2003; 129: 621-630Crossref PubMed Scopus (34) Google Scholar). CKAP2 is a p53 target gene that has been implicated in cell proliferation, genomic stability, and apoptosis, although the mechanism of its function remains largely unknown (23Jin Y. Murakumo Y. Ueno K. Hashimoto M. Watanabe T. Shimoyama Y. Ichihara M. Takahashi M. Cancer Sci. 2004; 95: 815-821Crossref PubMed Scopus (30) Google Scholar, 24Tsuchihara K. Lapin V. Bakal C. Okada H. Brown L. Hirota-Tsuchihara M. Zaugg K. Ho A. Itie-Youten A. Harris-Brandts M. Rottapel R. Richardson C.D. Benchimol S. Mak T.W. Cancer Res. 2005; 65: 6685-6691Crossref PubMed Scopus (40) Google Scholar, 25Jeon S.M. Choi B. Hong K.U. Kim E. Seong Y.S. Bae C.D. Park J. Biochem. Biophys. Res. Commun. 2006; 348: 222-228Crossref PubMed Scopus (23) Google Scholar). We showed that CKAP2 is a substrate of the APC/C-Cdh1, both in vitro and in vivo. The levels of CKAP2 fluctuated in the cell cycle, high in mitosis and low as cells exit into G1. Indeed, CKAP2 was degraded during mitotic exit in vivo in a Cdh1-dependent manner, as knockdown of Cdh1 stabilized the CKAP2 protein and increased its half-life. The APC/C-Cdh1 recognized a KEN-box in the N-terminal region of CKAP2, and mutations in this KEN-box abolished ubiquitination in vitro and stabilized the mutant protein in vivo. Furthermore, ectopic expression of CKAP2, either the wild-type or the mutant protein, led to a mitotic arrest with monopolar spindles containing highly bundled microtubules, indicating that the regulation of the CKAP2 protein levels is essential for normal mitotic progression. We concluded that CKAP2 is a physiological substrate of the APC/C whose mitotic degradation regulates its activity, and hence mitotic progression. A Genomic Approach to Identify Substrates of the APC/C—To identify candidate G2/M-induced APC/C substrates with expression patterns co-varying with those of known cell cycle regulators in tumor tissues, we used published microarray data-bases. Whitfield et al. (26Whitfield M.L. Sherlock G. Saldanha A.J. Murray J.I. Ball C.A. Alexander K.E. Matese J.C. Perou C.M. Hurt M.M. Brown P.O. Botstein D. Mol. Biol. Cell. 2002; 13: 1977-2000Crossref PubMed Scopus (1198) Google Scholar) performed genome-wide microarray analysis in HeLa cells across the cell cycle and identified 566 genes transcriptionally induced in G2 or in mitosis. Segal et al. (27Segal E. Friedman N. Koller D. Regev A. Nat. Genet. 2004; 36: 1090-1098Crossref PubMed Scopus (576) Google Scholar) analyzed co-expression profile of human genes among 1975 published microarrays derived from 22 different types of tumors. Based on statistic analyses of co-expression profiles, they organized human genes into different functional modules, each of which corresponds to a set of genes that act in concert to carry out a specific physiological function. Out of 577 functional modules identified, two modules function in the cell cycle regulation and consist of genes that co-vary (co-upregulate or co-downregulate) with known cell cycle regulators (27Segal E. Friedman N. Koller D. Regev A. Nat. Genet. 2004; 36: 1090-1098Crossref PubMed Scopus (576) Google Scholar). Thus, we analyzed the 566 G2/M-induced genes from Whitfield study (26Whitfield M.L. Sherlock G. Saldanha A.J. Murray J.I. Ball C.A. Alexander K.E. Matese J.C. Perou C.M. Hurt M.M. Brown P.O. Botstein D. Mol. Biol. Cell. 2002; 13: 1977-2000Crossref PubMed Scopus (1198) Google Scholar) for their co-variation with cell cycle genes identified by Segal et al. (27Segal E. Friedman N. Koller D. Regev A. Nat. Genet. 2004; 36: 1090-1098Crossref PubMed Scopus (576) Google Scholar). This allowed us to generate a candidate list of APC/C substrates. The full-length clones for these candidate genes were then purchased from Open Biosystems or requested from individual labs. The gene products were translated in vitro in reticulocyte lysates and assayed for ubiquitination by the APC/C-Cdh1 (8Zhao W.M. Fang G. J. Biol. Chem. 2005; 280: 33516-33524Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 13Fang G. Yu H. Kirschner M.W. Mol Cell. 1998; 2: 163-171Abstract Full Text Full Text PDF PubMed Scopus (426) Google Scholar). A detailed list of candidate genes will be reported in a separate study. We focus on the characterization of CKAP2 in this article. Plasmids and Antibodies—Human CKAP2 gene, a gift from Dr. Joobae Park (Sungkyunkwan University, Suwon, Korea), was subcloned into pCS2-FA and pCS2-eGFP-FA vectors. KEN-box mutation in CKAP2 was generated using the QuikChange site-directed mutagenesis kit (Stratagene). Histagged CKAP2 N-terminal domain (amino acids 1-404) was expressed in Escherichia coli, purified by nickel agarose under denaturing conditions, and used to immunize rabbits for production of antiserum. Anti-CKAP2 antibodies were subsequently purified against the antigen. In Vitro Ubiquitination Assays—Interphase and mitotic extracts were made from Xenopus eggs as described previously (14Fang G. Yu H. Kirschner M.W. Genes Dev. 1998; 12: 1871-1883Crossref PubMed Scopus (496) Google Scholar). Interphase extracts from Xenopus eggs were immunoprecipitated with anti-Cdc27 antibody-protein A beads for 2 h at 4 °C to purify interphase APC/C (iAPC/C) (8Zhao W.M. Fang G. J. Biol. Chem. 2005; 280: 33516-33524Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 13Fang G. Yu H. Kirschner M.W. Mol Cell. 1998; 2: 163-171Abstract Full Text Full Text PDF PubMed Scopus (426) Google Scholar). The iAPC/C beads were collected by centrifugation and washed five times in the XB buffer (10 mm HEPES-KOH, pH7.8, 100 mm KCl, 1 mm MgCl2, 0.1 mm CaCl2, 50 mm sucrose) containing 500 mm KCl and 0.5% Nonidet P-40, and two times in the XB buffer. Purified iAPC/C beads were then incubated with in vitro translated Cdh1 or Cdc20 for 1 h at 25 °C and then washed twice with the XB buffer. Ubiquitination reactions were initiated by mixing 35S-labeled substrate with E1 (50 μg/ml), E2 (50 μg/ml), ubiquitin (1.25 mg/ml), ubiquitin aldehyde (1 μm), and an energy regeneration mix (8Zhao W.M. Fang G. J. Biol. Chem. 2005; 280: 33516-33524Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 13Fang G. Yu H. Kirschner M.W. Mol Cell. 1998; 2: 163-171Abstract Full Text Full Text PDF PubMed Scopus (426) Google Scholar). Reactions were preformed at 25 °C and stopped at various times by addition of the SDS sample buffer. Samples from each time point were then analyzed by SDS-PAGE and Phosphorimager. In Vitro Degradation Assays—Interphase or mitotic Xenopus egg extracts, with or without prior incubation of in vitro translated Cdh1, were incubated with 35S-labeled substrates in the presence of ubiquitin (1.25 mg/ml), ubiquitin aldehyde (1 μm), and an energy regeneration mix at 30 °C, as described previously (13Fang G. Yu H. Kirschner M.W. Mol Cell. 1998; 2: 163-171Abstract Full Text Full Text PDF PubMed Scopus (426) Google Scholar). Samples from each time point were then analyzed by SDS-PAGE and Phosphorimager. Cell Culture, siRNAs, and Transfection—HeLa S3 and HeLa cells were cultured in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum (Invitrogen) and antibiotics. To determine the levels of the CKAP2 protein across the cell cycle, cells were synchronized either at the G1/S boundary by a double thymidine treatment or at prometaphase by a thymidine-nocodazole treatment (13Fang G. Yu H. Kirschner M.W. Mol Cell. 1998; 2: 163-171Abstract Full Text Full Text PDF PubMed Scopus (426) Google Scholar). siRNAs were synthesized by Dharmacon, Inc. Sequences targeted to CKAP2 were (A) 5′-GGATATGTCTTGCACTTATTT-3′, (B) 5′-GGACTACCATGGCAGAAGATT-3′, and (C) 5′-GAGGACAAGTTGCTTAATTTT-3′. siRNA against Cdh1 was 5′-GCAACGATGTGTCTCCCTATT-3′ and the control siRNA (siGL2) was 5′-CGTACGCGGAATACTTCGATT-3′. Short hairpin RNA (shRNA) against Cdh1 was synthesized by in vitro transcription with the following sequence: 5′-GGATTAACGAGAATGAGAAGT-3′. Control shRNA against GFP was 5′-GCAAGCTGACCCTGAAGTTC-3′. siRNAs were transfected into HeLa cells using DharmaFect 1 (Dharmacon, Inc.). For CKAP2, all three siRNAs gave similar degrees of knockdown and all three gave identical phenotypes, confirming the specificity of the knockdown. DNA transfection was performed using Effectene (Qiagen) or Lipofectamine 2000 (Invitrogen) as instructed by the manufacturers. In experiments with ectopic expression of CKAP2 and its KEN mutant, the levels of expressed proteins was about 15 folds compared with that of the endogenous CKAP2 protein. To determine the stability of the CKAP2 protein in vivo, transfected cells were arrested at prometaphase by a nocodazole treatment followed by release into fresh media in the presence of 10 μg/ml cycloheximide (8Zhao W.M. Fang G. J. Biol. Chem. 2005; 280: 33516-33524Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). Cells were harvested, lysed, and subjected to Western blot analysis. Immunofluorescence—To determine the localization of CKAP2, cells were cultured on poly-lysine-coated glass cover-slips overnight and then fixed with -20 °C methanol. After fixation, cells were permeabilized and blocked with PBS-BT (1× PBS, 0.1% Triton X-100, 3% bovine serum albumin) at the room temperature for 30 min. Coverslips were subsequently incubated in primary and secondary antibodies diluted in PBS-BT. Images were acquired with Openlab 5.0 (Improvision) under a Zeiss Axiovert 200M microscope using ×100 oil immersion lens. Deconvolved images were obtained using AutoDeblur v9.1 and AutoVisualizer v9.1 (AutoQuant Imaging). A Genomic Approach to Efficiently Identify Mitotic Substrates of APC/C—To identify novel mitotic substrates of APC/C, we selected candidate genes based on their gene expression profiles and then assayed their ability to be ubiquitinated by the APC/C in an in vitro reconstituted APC/C assay. Our genomic analysis was based on two predictions. First, we predicted that substrates that solely function in mitosis or cytokinesis are likely transcriptionally induced in G2 or in mitosis prior to their function. On the other hand, we expected that only a subset, but not all, of these G2/M-induced genes acts in mitosis and cytokinesis and therefore needed another criterion to select functionally important ones. Second, we predicted that expression of genes in the core mitosis and cytokinesis machinery tends to co-vary during tumorigenesis, as these regulators are likely to function as one module during tumor proliferation. It has been reported that 566 genes in human genome are induced in G2 or in mitosis in HeLa cells (26Whitfield M.L. Sherlock G. Saldanha A.J. Murray J.I. Ball C.A. Alexander K.E. Matese J.C. Perou C.M. Hurt M.M. Brown P.O. Botstein D. Mol. Biol. Cell. 2002; 13: 1977-2000Crossref PubMed Scopus (1198) Google Scholar). Thus, we searched in published microarray databases (27Segal E. Friedman N. Koller D. Regev A. Nat. Genet. 2004; 36: 1090-1098Crossref PubMed Scopus (576) Google Scholar, 28Rhodes D.R. Yu J. Shanker K. Deshpande N. Varambally R. Ghosh D. Barrette T. Pandey A. Chinnaiyan A.M. Neoplasia. 2004; 6: 1-6Crossref PubMed Google Scholar) for novel ones among these 566 genes that transcriptionally co-varied (co-induced or co-repressed) in hundreds of tumor tissues with known cell cycle regulators, such as cyclin A, cyclin B, Plk1, Aurora A, Aurora B, and Cdc20. From these analyses, we generated a list of G2/M-induced genes (the candidate list) whose expression co-varied with those of known cell cycle regulators across hundreds of tumor tissues. Next, we determined whether this list enriched known mitotic substrates of the APC/C. We analyzed 16 known substrates of the APC/C that have been characterized at the beginning of this study. These substrates included cyclin A, cyclin B, Plk1, Aurora A, Aurora B, Nek2, Cdc20, UbcH10, securin, GTSE1, anillin, TPX2, Cdc6, geminin, ribonucleotide reductase M2, and SnoN (3Harper J.W. Burton J.L. Solomon M.J. Genes Dev. 2002; 16: 2179-2206Crossref PubMed Scopus (424) Google Scholar,4Peters J.M. Mol. Cell. 2002; 9: 931-943Abstract Full Text Full Text PDF PubMed Scopus (778) Google Scholar,6Stewart S. Fang G. Cancer Res. 2005; 65: 8730-8735Crossref PubMed Scopus (125) Google Scholar, 7Stewart S. Fang G. Mol. Cell. Biol. 2005; 25: 10516-10527Crossref PubMed Scopus (82) Google Scholar, 8Zhao W.M. Fang G. J. Biol. Chem. 2005; 280: 33516-33524Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar,29Wan Y. Liu X. Kirschner M.W. Mol. Cell. 2001; 8: 1027-1039Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 30Stroschein S.L. Bonni S. Wrana J.L. Luo K. Genes Dev. 2001; 15: 2822-2836Crossref PubMed Google Scholar, 31Chabes A.L. Pfleger C.M. Kirschner M.W. Thelander L. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 3925-3929Crossref PubMed Scopus (136) Google Scholar). The first 15 substrates function in the cell cycle regulation (4Peters J.M. Mol. Cell. 2002; 9: 931-943Abstract Full Text Full Text PDF PubMed Scopus (778) Google Scholar) and, indeed, expression of all of them co-varied with known cell cycle genes (27Segal E. Friedman N. Koller D. Regev A. Nat. Genet. 2004; 36: 1090-1098Crossref PubMed Scopus (576) Google Scholar, 28Rhodes D.R. Yu J. Shanker K. Deshpande N. Varambally R. Ghosh D. Barrette T. Pandey A. Chinnaiyan A.M. Neoplasia. 2004; 6: 1-6Crossref PubMed Google Scholar). SnoN functions in cell signaling (29Wan Y. Liu X. Kirschner M.W. Mol. Cell. 2001; 8: 1027-1039Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 30Stroschein S.L. Bonni S. Wrana J.L. Luo K. Genes Dev. 2001; 15: 2822-2836Crossref PubMed Google Scholar) and, as expected, expression of SnoN did not co-vary with those of the cell cycle genes (27Segal E. Friedman N. Koller D. Regev A. Nat. Genet. 2004; 36: 1090-1098Crossref PubMed Scopus (576) Google Scholar, 28Rhodes D.R. Yu J. Shanker K. Deshpande N. Varambally R. Ghosh D. Barrette T. Pandey A. Chinnaiyan A.M. Neoplasia. 2004; 6: 1-6Crossref PubMed Google Scholar). Out of the 15 substrates involved in cell cycle regulation, the first 12 act in mitosis and cytokinesis (3Harper J.W. Burton J.L. Solomon M.J. Genes Dev. 2002; 16: 2179-2206Crossref PubMed Scopus (424) Google Scholar, 4Peters J.M. Mol. Cell. 2002; 9: 931-943Abstract Full Text Full Text PDF PubMed Scopus (778) Google Scholar, 6Stewart S. Fang G. Cancer Res. 2005; 65: 8730-8735Crossref PubMed Scopus (125) Google Scholar, 7Stewart S. Fang G. Mol. Cell. Biol. 2005; 25: 10516-10527Crossref PubMed Scopus (82) Google Scholar, 8Zhao W.M. Fang G. J. Biol. Chem. 2005; 280: 33516-33524Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). Interestingly, all 12 genes were transcriptionally induced in G2 or in mitosis (26Whitfield M.L. Sherlock G. Saldanha A.J. Murray J.I. Ball C.A. Alexander K.E. Matese J.C. Perou C.M. Hurt M.M. Brown P.O. Botstein D. Mol. Biol. Cell. 2002; 13: 1977-2000Crossref PubMed Scopus (1198) Google Scholar). The remaining three, Cdc6, geminin, and ribonucleotide reductase M2 function in S phase (9Petersen B.O. Wagener C. Marinoni F. Kramer E.R. Melixetian M. Denchi E.L. Gieffers C. Matteucci C. Peters J.M. Helin K. Genes Dev. 2000; 14: 2330-2343Crossref PubMed Scopus (233) Google Scholar, 10Li A. Blow J.J. EMBO J. 2005; 24: 395-404Crossref PubMed Scopus (115) Google Scholar, 11McGarry T.J. Kirschner M.W. Cell. 1998; 93: 1043-1053Abstract Full Text Full Text PDF PubMed Scopus (741) Google Scholar,31Chabes A.L. Pfleger C.M. Kirschner M.W. Thelander L. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 3925-3929Crossref PubMed Scopus (136) Google Scholar) and expression of all three peaked in S phase (26Whitfield M.L. Sherlock G. Saldanha A.J. Murray J.I. Ball C.A. Alexander K.E. Matese J.C. Perou C.M. Hurt M.M. Brown P.O. Botstein D. Mol. Biol. Cell. 2002; 13: 1977-2000Crossref PubMed Scopus (1198) Google Scholar). We concluded that our candidate list is highly enriched in mitotic substrates of the APC/C. Thus, we cloned the full-length genes from the candidate list, translated radioactively labeled proteins in vitro and then tested their ability to be ubiquitinated by the active APC/C-Cdh1 in a reconstituted assay (8Zhao W.M. Fang G. J. Biol. Chem. 2005; 280: 33516-33524Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 13Fang G. Yu H. Kirschner M.W. Mol Cell. 1998; 2: 163-171Abstract Full Text Full Text PDF PubMed Scopus (426) Google Scholar). From these experiments, we identified 9 new substrates of the APC/C: CKAP2, MELK, MKLP2, Ect2, INCENP, HSET and three novel proteins (Fig. 1; data not shown). We reported the characterization of CKAP2 as a substrate of APC/C in this article. CKAP2 Is a Substrate of the APC/C-Cdh1 in Vitro—We investigated whether CKAP2 is a substrate of the APC/C ubiquitin ligase. In vitro ubiquitination and degradation assays were used to directly analyze the role of APC/C in the stability of CKAP2. Our ubiquitination assay was a two-step process (8Zhao W.M. Fang G. J. Biol. Chem. 2005; 280: 33516-33524Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 13Fang G. Yu H. Kirschner M.W. Mol Cell. 1998; 2: 163-171Abstract Full Text Full Text PDF PubMed Scopus (426) Google Scholar). Interphase APC/C (iAPC/C) was first purified from Xenopus interphase extracts and activated by Cdc20 or Cdh1 that had been transcribed and translated in vitro. 35S-labeled CKAP2 was then incubated with active APC/C-Cdc20 or APC/C-Cdh1 and the kinetics of its ubiquitination assayed by SDS-PAGE. CKAP2 was efficiently ubiquitinated by APC/C-Cdh1, but not by APC/C-Cdc20 (Fig. 1A). The CKAP2-ub conjugates migrated as a smear above the input CKAP2 substrate. The APC/C-Cdc20 used in our assay was fully active, as it quantitatively ubiquitinated securin (Fig. 1B). The extent of ubiquitination on securin by APC/C-Cdc20 and APC/C-Cdh1 was so efficient that the heterogeneous securin-ub conjugates migrated as a smear across the whole lane, barely detectable in 15-60 min samples. To further confirm that ubiquitination of CKAP2 is APC/C-Cdh1-dependent, the recombinant Emi1, an inhibitor of APC/C (18Reimann J.D. Freed E. Hsu J.Y. Kramer E.R. Peters J.M. Jackson P.K. Cell. 2001; 105: 645-655Abstract Full Text Full Text PDF PubMed Scopus (310) Google Scholar), was incubated with APC/C and Cdh1 prior to the addition of the CKAP2 substrate. As expected, Emi1 efficiently blocked the ubiquitination of CKAP2 by APC/C-Cdh1 (Fig. 1C). Xenopus extracts are a powerful system to analyze the protein stability in a cell cycle dependent manner (13Fang G. Yu H. Kirschner M.W. Mol Cell. 1998; 2: 163-171Abstract Full Text Full Text PDF PubMed Scopus (426) Google Scholar, 14Fang G. Yu H. Kirschner M.W. Genes Dev. 1998; 12: 1871-1883Crossref PubMed Scopus (496) Google Scholar, 32Fang G. Yu H. Kirschner M.W. Philos Trans R Soc Lond. B Biol. Sci. 1999; 354: 1583-1590Crossref PubMed Scopus (74) Google Scholar). Although interphase extracts did not have any APC/C activity, addition of in vitro translated Cdh1 efficiently activated APC/C, leading to the degradation of 35S-labeled securin (Fig. 2B). The mitotic extracts had active APC/C-Cdc20, which targeted securin for degradation (Fig. 2B). When incubated with 35S-labeled CKAP2, Cdh1-a" @default.
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• W2078702698 title "CKAP2 Is a Spindle-associated Protein Degraded by APC/C-Cdh1 during Mitotic Exit" @default.
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