Global Analysis of Cdc14 Dephosphorylation Sites Reveals Essential Regulatory Role in Mitosis and Cytokinesis
2013; Elsevier BV; Volume: 13; Issue: 2 Linguagem: Inglês
10.1074/mcp.m113.032680
ISSN1535-9484
AutoresLi‐Ting Kao, Yi‐Ting Wang, Yu‐Chen Chen, Shun‐Fu Tseng, Jia-Cin Jhang, Yu‐Ju Chen, Shu‐Chun Teng,
Tópico(s)Photosynthetic Processes and Mechanisms
ResumoDegradation of the M phase cyclins triggers the exit from M phase. Cdc14 is the major phosphatase required for the exit from the M phase. One of the functions of Cdc14 is to dephosphorylate and activate the Cdh1/APC/C complex, resulting in the degradation of the M phase cyclins. However, other crucial targets of Cdc14 for mitosis and cytokinesis remain to be elucidated. Here we systematically analyzed the positions of dephosphorylation sites for Cdc14 in the budding yeast Saccharomyces cerevisiae. Quantitative mass spectrometry identified a total of 835 dephosphorylation sites on 455 potential Cdc14 substrates in vivo. We validated two events, and through functional studies we discovered that Cdc14-mediated dephosphorylation of Smc4 and Bud3 is essential for proper mitosis and cytokinesis, respectively. These results provide insight into the Cdc14-mediated pathways for exiting the M phase. Degradation of the M phase cyclins triggers the exit from M phase. Cdc14 is the major phosphatase required for the exit from the M phase. One of the functions of Cdc14 is to dephosphorylate and activate the Cdh1/APC/C complex, resulting in the degradation of the M phase cyclins. However, other crucial targets of Cdc14 for mitosis and cytokinesis remain to be elucidated. Here we systematically analyzed the positions of dephosphorylation sites for Cdc14 in the budding yeast Saccharomyces cerevisiae. Quantitative mass spectrometry identified a total of 835 dephosphorylation sites on 455 potential Cdc14 substrates in vivo. We validated two events, and through functional studies we discovered that Cdc14-mediated dephosphorylation of Smc4 and Bud3 is essential for proper mitosis and cytokinesis, respectively. These results provide insight into the Cdc14-mediated pathways for exiting the M phase. All cells proliferate following a fixed, highly coordinated cycle. Mitosis especially requires elaborate coordination for proper chromosome segregation, mitotic spindle disassembly, and cytokinesis. Much of this activity is facilitated by numerous, diverse phosphorylation and dephosphorylation signals that orchestrate the precise progression of M phase. Prior to mitosis, sister chromatids resulting from DNA replication during S phase are held together by the cohesion complex. Then, during prophase, chromosomes are condensed by the condensin (Smc2/4) complex (1Hirano T. Mitchison T.J. A heterodimeric coiled-coil protein required for mitotic chromosome condensation in vitro.Cell. 1994; 79: 449-458Abstract Full Text PDF PubMed Scopus (431) Google Scholar) and microtubules are remodeled to form the mitotic spindle (2Haase S.B. Winey M. Reed S.I. Multi-step control of spindle pole body duplication by cyclin-dependent kinase.Nat. Cell Biol. 2001; 3: 38-42Crossref PubMed Scopus (76) Google Scholar). 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At interphase, Cdc14 is a subunit of the mitotic exit network (14Bardin A.J. Visintin R. Amon A. A mechanism for coupling exit from mitosis to partitioning of the nucleus.Cell. 2000; 102: 21-31Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar, 15Guacci V. Hogan E. Koshland D. Chromosome condensation and sister chromatid pairing in budding yeast.J. Cell Biol. 1994; 125: 517-530Crossref PubMed Scopus (279) Google Scholar, 16Jaspersen S.L. Charles J.F. Tinker-Kulberg R.L. Morgan D.O. A late mitotic regulatory network controlling cyclin destruction in.Saccharomyces cerevisiae. Mol. Biol. Cell. 1998; 9: 2803-2817Crossref Scopus (263) Google Scholar, 17Jaspersen S.L. Morgan D.O. Cdc14 activates cdc15 to promote mitotic exit in budding yeast.Curr. Biol. 2000; 10: 615-618Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar), which usually localizes to the nucleolus. 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Cfi1 prevents premature exit from mitosis by anchoring Cdc14 phosphatase in the nucleolus.Nature. 1999; 398: 818-823Crossref PubMed Scopus (487) Google Scholar), leading to exit from mitosis. In addition to this essential role in late M phrase, Cdc14 substrates have also been identified in other stages of the cell cycle (19Akiyoshi B. Biggins S. Cdc14-dependent dephosphorylation of a kinetochore protein prior to anaphase in.Saccharomyces cerevisiae. Genetics. 2010; 186: 1487-1491Crossref Scopus (15) Google Scholar). cyclin-dependent kinase sodium dodecyl sulfate-polyacrylamide gel electrophoresis. cyclin-dependent kinase sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Cdc14 putatively regulates 27 proteins (19Akiyoshi B. Biggins S. Cdc14-dependent dephosphorylation of a kinetochore protein prior to anaphase in.Saccharomyces cerevisiae. Genetics. 2010; 186: 1487-1491Crossref Scopus (15) Google Scholar, 20Mocciaro A. Schiebel E. 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Cell Biol. 2007; 177: 981-993Crossref PubMed Scopus (122) Google Scholar), suggesting that spurious relationships cannot be ruled out. Also, experiments have not been carried out to demonstrate whether these modifications entail direct or indirect regulation. Therefore, our understanding of Cdc14 function and regulation during mitosis in metazoans is incomplete. Conceivably, Cdc14 may regulate many more substrates involved in aspects of chromosome condensation and cytokinesis. To examine this possibility we performed a systematic phosphoproteomic screen to identify new in vivo pathways regulated by Cdc14. Using this approach, we identified both known and potentially novel substrates of Cdc14, as well as their dephosphorylation sites. Many potentially novel substrates are physically associated with Cdc14 in public databases. We also provide biochemical evidence for direct dephosphorylation of the substrates, characterize the specificity of dephosphorylation in two substrates, Smc4 and Bud3, and further study their regulation and critical role in mitosis and cytokinesis. All yeast operations were performed using standard methods. Strains used in this study are listed in supplemental Table S1. Strains were constructed and analyzed via standard genetic methods. Cells were grown in rich medium (YEPD: 1% yeast extract, 2% bacto-peptone, 2% dextrose) or, for plasmid selection, in synthetic complete medium (0.67% yeast nitrogen base; 2% glucose, raffinose, or galactose). S288C and cdc28-4 (26Lorincz A.T. Reed S.I. Sequence analysis of temperature-sensitive mutations in the Saccharomyces cerevisiae gene.CDC28. Mol. Cell. Biol. 1986; 6: 4099-4103Crossref PubMed Scopus (46) Google Scholar) strains were used to detect phosphorylation and Y300 and cdc14-1 strains were used to detect dephosphorylation in vivo. CDC14, BUD3, and SMC4 were PCR-amplified and cloned into pRS306. Point mutations were introduced into plasmids using QuickChange site-directed mutagenesis (Stratagene, Santa Clara, CA). The cdc14-1 strain was constructed by insertion of pRS306-cdc14-D323G following 5-FOA-resistant selection. To generate chromosomal mutations of BUD3 and SMC4, pRS306-bud3 and pRS306-smc4 were linearized by EcoRI and HpaI, respectively, and transformed into the wild-type cells. The URA3 pop-out mutants were selected from the 5-FOA-resistant transformants. The mutations were checked by PCR and sequencing. pGH-bud3Cter (1534–1636) was constructed by ligating the BamHI/XhoI-treated fragment containing amino acids 1534–1636 of Bud3 into the BamHI/XhoI-digested pGH. Details of plasmid constructions are available upon request. Chromosomal tagging was constructed as previously described (27Schneider B.L. Seufert W. Steiner B. Yang Q.H. Futcher A.B. 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Wang Y.T. Chen Y.R. Lai C.Y. Lin P.Y. Pan K.T. Chen J.Y. Khoo K.H. Chen Y.J. Immobilized metal affinity chromatography revisited: pH/acid control toward high selectivity in phosphoproteomics.J. Proteome Res. 2008; 7: 4058-4069Crossref PubMed Scopus (109) Google Scholar). First, the column was capped at one end with a 0.5-μm frit disk enclosed in a stainless steel column-end fitting. The nickel-nitrilotriacetic acid resin from the spin column (Qiagen, Hilden, Germany) was packed into a 10-cm microcolumn (500-μm inner diameter PEEK column, Upchurch Scientific/Rheodyne, Oak Harbor, WA). Automated purification of phosphopeptides was performed using an Ultra-Performance LCTM system (Waters Corp., Milford, MA) with the flow rate at 13 μl/min. First, the Ni2+ ions were removed with 100 μl 50 mm EDTA in 1 m NaCl. The nitrilotriacetic acid resin was activated by 100 μl 0.2 m FeCl3 and equilibrated with loading buffer for 15 min before sample loading. The loading/condition buffer was 6% (v/v) acetic acid (pH 3.0). The peptide samples from trypsin digestion were reconstituted in loading buffer and loaded into an activated immobilized metal affinity chromatography column pre-equilibrated with the same loading buffer for 12 min. The unbound peptides were then removed with 100 μl washing solution consisting of 75% (v/v) loading buffer and 25% (v/v) acetonitrile, followed by equilibration with loading buffer for 15 min. Finally, the bound peptides were eluted with 100 μl of 200 mm NH4H2PO4 (pH 4.4). Eluted peptide samples were dried and reconstituted in 0.1% (v/v) TFA (40 μl) for further desalting and concentration using ZipTipsTM (Millipore, Bedford, CA). Purified phosphopeptides were reconstituted in buffer A (0.1% formic acid in H2O) and analyzed on an LTQ-Orbitrap (Thermo Electron, Bremen, Germany) in triplicate. For LTQ-Orbitrap analysis, LC-MS/MS was performed on an Agilent 1100 series HPLC (Agilent, Waldbronn, Germany) with a micro-T for flow splitting connected to an LTQ-Orbitrap XL hybrid mass spectrometer. Peptides were loaded onto a 25 cm × 75 μm fused-silica capillary column packed in-house with C18 gel (5 μm, Nucleosil 120–5 C18, Macherey-Nagel, GmbH & Co. KG, Düren, Germany) and separated using a segmented gradient over 100 min from 2% to 80% buffer B. Survey full-scan MS spectra were acquired in the Orbitrap (m/z 350–1600) with the resolution set to 60,000 at m/z 400 and the automatic gain control target at 106. The 10 most intense ions were sequentially isolated for collision-induced dissociation MS/MS fragmentation and detection in the linear ion trap (automatic gain control target at 7000) with previously selected ions dynamically excluded for 90 s. To improve the fragmentation spectra of the phosphopeptides, "multistage activation" at 97.97, 48.99, and 32.66 Thompson (Th) relative to the precursor ion was enabled in all MS/MS events. All the measurements in the Orbitrap were performed with the lock mass option for internal calibration. Raw MS/MS data from the LTQ-Orbitrap were transformed to msm files using the software RAW2MSM (version 1.1) (32Olsen J.V. de Godoy L.M. Li G. Macek B. Mortensen P. Pesch R. Makarov A. Lange O. Horning S. Mann M. Parts per million mass accuracy on an Orbitrap mass spectrometer via lock mass injection into a C-trap.Mol. Cell. Proteomics. 2005; 4: 2010-2021Abstract Full Text Full Text PDF PubMed Scopus (1241) Google Scholar). The msm files were searched using Mascot (version 2.2.1) against the Swiss-Prot Saccharomyces cerevisiae (baker's yeast) database (version 54.2, 6493 sequences) with the following exceptions: only tryptic peptides with up to two missed cleavage sites were allowed, the fragment ion mass tolerance was set at 10 ppm, and the parent ion tolerance was set at 0.6 Da. Phosphorylation (STY) and oxidation (M) were specified as variable modifications. Peptides were considered identified if their Mascot individual ion score was greater than 20 (p < 0.05). The false discovery rates for Orbitrap data were determined with a Mascot score greater than 20 (p < 0.05) in this study. All of the raw datasets, peak lists, spectra of identified phosphopeptides, and identification results have been deposited in the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository (33Vizcaino J.A. Cote R.G. Csordas A. Dianes J.A. Fabregat A. Foster J.M. Griss J. Alpi E. 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To increase correct assignment, the detected peptide peaks were validated by means of SCI validation using three criteria: (i) signal-to-noise ratio > 3, (ii) accurate charge state, and (iii) correct isotope pattern. To calculate the relative peptide abundance, the tool performs reconstruction of extracted ion chromatography and calculates the area of the extracted ion chromatograph. The fold-change of a given peptide was calculated based on the ratio of relative peptide abundance between different samples. Finally, the quantitation result for each phosphopeptide was manually checked. The analysis of identified peptides is presented in supplemental Tables S2 and S3. Gene Ontology analysis was conducted using an online tool available at the Saccharomyces Genome Database website (version 8.03) (36Boyle E.I. Weng S. Gollub J. Jin H. Botstein D. Cherry J.M. Sherlock G. 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Pre-immune serums were collected before boost. Injection was conducted every 4 weeks, and blood samples were collected every 2 weeks. Blood samples were incubated at 37 °C for 30 min, and serum and blood cells were separated via high-speed centrifugation. Clarified serum was incubated at 56 °C for 30 min to remove complements. The specificity of antibodies was verified by means of peptide dot blot analysis. Whole cell proteins were extracted via trichloroacetic acid precipitation and resolved via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Smc4 was detected with a Myc antibody (Roche) and the phospho-antibody, and Bud3 was detected with an HA antibody (Covance, Princeton, NJ, monoclonal antibody, HA.11) and the phospho-antibody. Cells were also stained with propidium iodide, and the cell cycle analysis was performed using a flow cytometer (FACSCalibur 200, BD Biosciences). 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After immunoprecipitation-kinase assay, phosphorylated Bud3 and Smc4 were desalted and changed to phosphatase buffer by Microcon 10K (Millipore) for further phosphatase assay as described elsewhere (22Zhai Y. Yung P.Y. Huo L. Liang C. Cdc14p resets the competency of replication licensing by dephosphorylating multiple initiation proteins during mitotic exit in budding yeast.J. Cell Sci. 2010; 123: 3933-3943Crossref PubMed Scopus (25) Google Scholar). β-galactosidase assays were performed as modified from Miller's protocol (38Guarente L. Yeast promoters and lacZ fusions designed to study expression of cloned genes in yeast.Methods Enzymol. 1983; 101: 181-191Crossref PubMed Scopus (873) Google Scholar, 39Miller J.H. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1972Google Scholar). pEG202-LexA-CLB2, pJG4–5-bud3Cter, and their derivatives were transformed into strain EGY191. 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