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• W2146453026 abstract "Polo-like kinases regulate many aspects of mitotic and meiotic progression from yeast to man. In early mitosis, mammalian Polo-like kinase 1 (Plk1) controls centrosome maturation, spindle assembly, and microtubule attachment to kinetochores. However, despite the essential and diverse functions of Plk1, the full range of Plk1 substrates remains to be explored. To investigate the Plk1-dependent phosphoproteome of the human mitotic spindle, we combined stable isotope labeling by amino acids in cell culture with Plk1 inactivation or depletion followed by spindle isolation and mass spectrometry. Our study identified 358 unique Plk1-dependent phosphorylation sites on spindle proteins, including novel substrates, illustrating the complexity of the Plk1-dependent signaling network. Over 100 sites were validated by in vitro phosphorylation of peptide arrays, resulting in a broadening of the Plk1 consensus motif. Collectively, our data provide a rich source of information on Plk1-dependent phosphorylation, Plk1 docking to substrates, the influence of phosphorylation on protein localization, and the functional interaction between Plk1 and Aurora A on the early mitotic spindle. Polo-like kinases regulate many aspects of mitotic and meiotic progression from yeast to man. In early mitosis, mammalian Polo-like kinase 1 (Plk1) controls centrosome maturation, spindle assembly, and microtubule attachment to kinetochores. However, despite the essential and diverse functions of Plk1, the full range of Plk1 substrates remains to be explored. To investigate the Plk1-dependent phosphoproteome of the human mitotic spindle, we combined stable isotope labeling by amino acids in cell culture with Plk1 inactivation or depletion followed by spindle isolation and mass spectrometry. Our study identified 358 unique Plk1-dependent phosphorylation sites on spindle proteins, including novel substrates, illustrating the complexity of the Plk1-dependent signaling network. Over 100 sites were validated by in vitro phosphorylation of peptide arrays, resulting in a broadening of the Plk1 consensus motif. Collectively, our data provide a rich source of information on Plk1-dependent phosphorylation, Plk1 docking to substrates, the influence of phosphorylation on protein localization, and the functional interaction between Plk1 and Aurora A on the early mitotic spindle. During mitosis, multiple processes, such as mitotic entry, spindle assembly, chromosome segregation, and cytokinesis, must be carefully coordinated to ensure the error-free distribution of chromosomes into the newly forming daughter cells. The physical separation of the chromosomes to opposite poles of the cell is driven by the mitotic spindle, a proteinaceous and highly dynamic microtubule (MT) 1The abbreviations used are:MTmicrotubulePlkPolo-like kinaseKTkinetochorePBDpolo box domainSILACstable isotope labeling by amino acids in cell cultureTALZK-thiazolidinoneMAmonastrolLTQlinear triple quadrupoleIPIInternational Protein IndexFDRfalse discovery rateDICdifferential interference contrastMAPMT-associated proteinNPCnuclear pore complexNUPnucleoporinCENPcentromere proteinBis-Tris2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diolH/Lheavy/lightTettetracyclineINCENPinner centromere proteinMgcRacGAPmale germ cell Rac GTPase-activating proteinCRESTcalcinosis, raynaud phenomenon, esophageal dismotility, sclerodactyly, telangectlasiaDAPI 4′6-diamidino-2-phenylindoleshRNAsmall hairpin RNA.-based macromolecular machine. Spindle assembly begins early in mitosis and is completed when the bipolar attachment of microtubules to kinetochore (KT) pairs is achieved (1.Cheeseman I.M. Desai A. Molecular architecture of the kinetochore-microtubule interface.Nat. Rev. Mol. Cell Biol. 2008; 9: 33-46Crossref PubMed Scopus (700) Google Scholar, 2.Musacchio A. Salmon E.D. The spindle-assembly checkpoint in space and time.Nat. Rev. Mol. Cell Biol. 2007; 8: 379-393Crossref PubMed Scopus (1739) Google Scholar). Polo-like kinase 1 (Plk1), a serine/threonine-specific kinase first identified in Drosophila (3.Llamazares S. Moreira A. Tavares A. Girdham C. Spruce B.A. Gonzalez C. Karess R.E. Glover D.M. Sunkel C.E. polo encodes a protein kinase homolog required for mitosis in Drosophila.Genes Dev. 1991; 5: 2153-2165Crossref PubMed Scopus (337) Google Scholar), is one of the key regulators of this essential mitotic process and has therefore attracted much attention (4.Barr F.A. Silljé H.H. Nigg E.A. Polo-like kinases and the orchestration of cell division.Nat. Rev. Mol. Cell Biol. 2004; 5: 429-440Crossref PubMed Scopus (913) Google Scholar, 5.Petronczki M. Lénárt P. Peters J.M. Polo on the rise—from mitotic entry to cytokinesis with Plk1.Dev. Cell. 2008; 14: 646-659Abstract Full Text Full Text PDF PubMed Scopus (408) Google Scholar, 6.Archambault V. Glover D.M. Polo-like kinases: conservation and divergence in their functions and regulation.Nat. Rev. Mol. Cell Biol. 2009; 10: 265-275Crossref PubMed Scopus (497) Google Scholar). In agreement with its diverse functions, the localization of Plk1 during mitosis is dynamic. Plk1 first associates with centrosomes in prophase before it localizes to spindle poles and KTs in prometaphase and metaphase. During anaphase, Plk1 is recruited to the central spindle and finally accumulates at the midbody during telophase. Proteomics studies using oriented peptide libraries have shown that two so-called polo boxes at the C-terminal end of Plk1, the polo box domain (PBD), are crucial for the localization of this kinase to cellular structures (7.Elia A.E. Cantley L.C. Yaffe M.B. Proteomic screen finds pSer/pThr-binding domain localizing Plk1 to mitotic substrates.Science. 2003; 299: 1228-1231Crossref PubMed Scopus (577) Google Scholar, 8.Elia A.E. Rellos P. Haire L.F. Chao J.W. Ivins F.J. Hoepker K. Mohammad D. Cantley L.C. Smerdon S.J. Yaffe M.B. The molecular basis for phosphodependent substrate targeting and regulation of Plks by the Polo-box domain.Cell. 2003; 115: 83-95Abstract Full Text Full Text PDF PubMed Scopus (611) Google Scholar). This domain binds to specific phosphorylated sequence motifs that are created by other priming kinases or are self-primed by Plk1 itself, thus providing an efficient mechanism to regulate localization and substrate selectivity in time and space (9.Kang Y.H. Park J.E. Yu L.R. Soung N.K. Yun S.M. Bang J.K. Seong Y.S. Yu H. Garfield S. Veenstra T.D. Lee K.S. Self-regulated Plk1 recruitment to kinetochores by the Plk1-PBIP1 interaction is critical for proper chromosome segregation.Mol. Cell. 2006; 24: 409-422Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar, 10.Lowery D.M. Clauser K.R. Hjerrild M. Lim D. Alexander J. Kishi K. Ong S.E. Gammeltoft S. Carr S.A. Yaffe M.B. Proteomic screen defines the Polo-box domain interactome and identifies Rock2 as a Plk1 substrate.EMBO J. 2007; 26: 2262-2273Crossref PubMed Scopus (201) Google Scholar, 11.Neef R. Preisinger C. Sutcliffe J. Kopajtich R. Nigg E.A. Mayer T.U. Barr F.A. Phosphorylation of mitotic kinesin-like protein 2 by polo-like kinase 1 is required for cytokinesis.J. Cell Biol. 2003; 162: 863-875Crossref PubMed Scopus (259) Google Scholar). microtubule Polo-like kinase kinetochore polo box domain stable isotope labeling by amino acids in cell culture ZK-thiazolidinone monastrol linear triple quadrupole International Protein Index false discovery rate differential interference contrast MT-associated protein nuclear pore complex nucleoporin centromere protein 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol heavy/light tetracycline inner centromere protein male germ cell Rac GTPase-activating protein calcinosis, raynaud phenomenon, esophageal dismotility, sclerodactyly, telangectlasia 6-diamidino-2-phenylindole small hairpin RNA. Despite the pleiotropic and critical functions of Plk1 during mitosis, only a limited number of target proteins and phosphorylation sites on substrates have so far been identified or studied in detail (4.Barr F.A. Silljé H.H. Nigg E.A. Polo-like kinases and the orchestration of cell division.Nat. Rev. Mol. Cell Biol. 2004; 5: 429-440Crossref PubMed Scopus (913) Google Scholar, 5.Petronczki M. Lénárt P. Peters J.M. Polo on the rise—from mitotic entry to cytokinesis with Plk1.Dev. Cell. 2008; 14: 646-659Abstract Full Text Full Text PDF PubMed Scopus (408) Google Scholar, 6.Archambault V. Glover D.M. Polo-like kinases: conservation and divergence in their functions and regulation.Nat. Rev. Mol. Cell Biol. 2009; 10: 265-275Crossref PubMed Scopus (497) Google Scholar, 12.Plyte S. Musacchio A. PLK1 inhibitors: setting the mitotic death trap.Curr. Biol. 2007; 17: R280-283Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). The difficulties in identification of bona fide Plk1 substrates stem from the low abundance of some substrates, technical limitations for determining in vivo phosphorylation sites, the requirement for Plk1 localization for recognition of some substrates, and the possibility that Plk1 may phosphorylate a broader consensus motif than determined previously (13.Nakajima H. Toyoshima-Morimoto F. Taniguchi E. Nishida E. Identification of a consensus motif for Plk (Polo-like kinase) phosphorylation reveals Myt1 as a Plk1 substrate.J. Biol. Chem. 2003; 278: 25277-25280Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar). Recent developments in mass spectrometry (MS)-based proteomics have allowed the identification of a large number of in vivo phosphorylation sites from complex samples (14.Schreiber T.B. Mäusbacher N. Breitkopf S.B. Grundner-Culemann K. Daub H. Quantitative phosphoproteomics—an emerging key technology in signal-transduction research.Proteomics. 2008; 8: 4416-4432Crossref PubMed Scopus (56) Google Scholar). However, the nature of the kinase(s) responsible for most of these phosphorylation events is still unclear, and the assignment of phosphorylation sites to individual kinases remains a challenging task. Previously, we explored the human mitotic spindle by MS and successfully identified a large number of novel spindle proteins and phosphorylation sites (15.Nousiainen M. Silljé H.H. Sauer G. Nigg E.A. Körner R. Phosphoproteome analysis of the human mitotic spindle.Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 5391-5396Crossref PubMed Scopus (272) Google Scholar, 16.Sauer G. Körner R. Hanisch A. Ries A. Nigg E.A. Silljé H.H. Proteome analysis of the human mitotic spindle.Mol. Cell. Proteomics. 2005; 4: 35-43Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). Now, the development of quantitative methods to monitor in vivo phosphorylation changes in complex samples (17.Olsen J.V. Blagoev B. Gnad F. Macek B. Kumar C. Mortensen P. Mann M. Global, in vivo, and site-specific phosphorylation dynamics in signaling networks.Cell. 2006; 127: 635-648Abstract Full Text Full Text PDF PubMed Scopus (2833) Google Scholar, 18.Dephoure N. Zhou C. Villén J. Beausoleil S.A. Bakalarski C.E. Elledge S.J. Gygi S.P. A quantitative atlas of mitotic phosphorylation.Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 10762-10767Crossref PubMed Scopus (1262) Google Scholar, 19.Olsen J.V. Vermeulen M. Santamaria A. Kumar C. Miller M.L. Jensen L.J. Gnad F. Cox J. Jensen T.S. Nigg E.A. Brunak S. Mann M. Quantitative phosphoproteomics reveals widespread full phosphorylation site occupancy during mitosis.Sci. Signal. 2010; 3: ra3Crossref PubMed Scopus (1163) Google Scholar) represents a unique opportunity to address the role of individual kinases in spindle function. To study Plk1 function at the mitotic spindle, we combined quantitative proteomics using stable isotope labeling by amino acids in cell culture (SILAC) (20.Ong S.E. Blagoev B. Kratchmarova I. Kristensen D.B. Steen H. Pandey A. Mann M. Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics.Mol. Cell. Proteomics. 2002; 1: 376-386Abstract Full Text Full Text PDF PubMed Scopus (4625) Google Scholar) with the isolation of human mitotic spindles and phosphopeptide enrichment. To expand the experimental coverage of Plk1 substrates and gain further insight into direct and indirect functions of Plk1, we compared the phosphoproteomes of mitotic spindles isolated from cells lacking Plk1 activity with spindles from cells with fully active kinase. Two independent approaches were used to interfere with Plk1 activity: protein depletion using an inducible small hairpin (shRNA) cell line and selective inhibition of the kinase by the small molecule inhibitor ZK-thiazolidinone (TAL) (21.Santamaria A. Neef R. Eberspächer U. Eis K. Husemann M. Mumberg D. Prechtl S. Schulze V. Siemeister G. Wortmann L. Barr F.A. Nigg E.A. Use of the novel Plk1 inhibitor ZK-thiazolidinone to elucidate functions of Plk1 in early and late stages of mitosis.Mol. Biol. Cell. 2007; 18: 4024-4036Crossref PubMed Scopus (166) Google Scholar). Phosphorylation sites found to be down-regulated after Plk1 inhibition/depletion were subsequently validated using in vitro phosphorylation of synthetic peptide arrays. This approach identified many candidate Plk1 substrates, allowed confirmation of direct phosphorylation by Plk1 of more than 100 sites identified in vivo, and suggested a broader phosphorylation consensus motif for this kinase. Collectively, our data set provides a rich resource for in-depth studies on the spindle-associated Plk1-dependent phosphoproteome. This is illustrated by selective follow-up studies in which we validated the Plk1-dependent localization of substrates to centrosomes and kinetochores. In particular, using a phosphospecific antibody, we confirmed Plk1-dependent CENP-F phosphorylation in vivo and demonstrated that CENP-F localization to kinetochores depends on Plk1 kinase activity. Furthermore, we identified several Aurora A-dependent phosphorylation events that are regulated by Plk1, supporting the emerging view of an intimate functional relationship between Plk1 and Aurora A kinase (22.Eckerdt F. Maller J.L. Kicking off the polo game.Trends Biochem. Sci. 2008; 33: 511-513Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar, 23.Macurek L. Lindqvist A. Medema R.H. Aurora-A and hBora join the game of Polo.Cancer Res. 2009; 69: 4555-4558Crossref PubMed Scopus (46) Google Scholar). For SILAC, HeLa S3 cells were grown in Dulbecco's modified Eagle's medium (DMEM) deficient in amino acids arginine and lysine and supplemented with 5% dialyzed FCS, 100 units/ml penicillin, 100 μg/ml streptomycin, and either unlabeled arginine·HCl and lysine·HCl (SILAC light) or l-[U-13C6,15N4]arginine·HCl and l-[U-13C6,15N2]lysine· HCl (SILAC heavy) (Cambridge Isotope Laboratories) at concentrations of 42 (arginine) and 72 μg/ml (lysine). Cells were grown at 37 °C in a humidified incubator with 5% atmospheric CO2. Cells were adapted to the appropriate SILAC medium for at least six passages to achieve complete incorporation of the isotope-labeled amino acids. For large scale mitotic spindle isolation, each population of HeLa S3 labeled cells was propagated to five triple flasks with a total surface of 500 cm2. Cells were first presynchronized with thymidine (2 mm) for 20 h, then washed twice with PBS, and released from the thymidine block into TAL or monastrol (MA)-containing medium (1 or 150 μm, respectively) or by induction with 1 μg/ml tetracycline for 36 h in the case of the inducible cell lines. Taxol-stabilized mitotic spindles (including kinetochores and centrosomes) were isolated as described previously (24.Silljé H.H. Nigg E.A. Purification of mitotic spindles from cultured human cells.Methods. 2006; 38: 25-28Crossref PubMed Scopus (17) Google Scholar). Enriched spindle proteins were separated by SDS-PAGE and in-gel (25.Shevchenko A. Wilm M. Vorm O. Mann M. Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels.Anal. Chem. 1996; 68: 850-858Crossref PubMed Scopus (7831) Google Scholar) digested with trypsin. In each experiment, about 400 μg of the spindle fraction was loaded to one NuPAGE Bis-Tris gel and run for 50 min using a 200-V/100-mA program. Gels were stained with a 1:1 mixture of 0.2% Coomassie Blue in methanol (MeOH) and 20% acetic acid for 40 min and destained using a mixture of 30% MeOH and 10% acetic acid for 15 min, 10% acetic acid for 1 h, and 2% acetic acid overnight at 4 °C. Gels were cut into 20 slices each. After reduction and alkylation, proteins were digested by 15 ng/μl trypsin for 16 h. Digested peptides were extracted with 30% ACN and 5% formic acid and dried. Phosphorylated peptides were selectively enriched by titanium dioxide beads with lactic acid as a modifier (26.Jensen S.S. Larsen M.R. Evaluation of the impact of some experimental procedures on different phosphopeptide enrichment techniques.Rapid Commun. Mass Spectrom. 2007; 21: 3635-3645Crossref PubMed Scopus (234) Google Scholar). A piece of C8 material was plugged at the constricted end of a GELoader tip, and about 3 mg of titanium dioxide beads was transferred to each of the microcolumns. The microcolumns were washed with 40 μl of 0.3 μg/μl lactic acid in a mixture of 80% ACN and 0.2% TFA. Digested peptides were redissolved in 0.3 μg/μl lactic acid in a mixture of 80% ACN and 2% TFA and applied to microcolumns with a slow flow rate, allowing phosphorylated peptides to bind to the titanium dioxide beads. The microcolumns were subsequently washed with 40 μl of 0.3 μg/μl lactic acid in a mixture of 80% ACN and 0.2% TFA and 40 μl of a mixture of 80% ACN and 0.2% TFA. Phosphorylated peptides were eluted with 40 μl of 0.6% NH4OH and 40 μl of a mixture of 80% ACN and 0.2% TFA. The eluates were dried and redissolved in 0.5% formic acid for LC-MS/MS analysis. The flow-through fractions were desalted with C18 reversed-phase material prior to LC-MS/MS for protein expression level measurement. Briefly, a piece of C18 material was plugged into a GELoader tip and washed with 20 μl of 2-propanol and 20 μl of 5% formic acid. The dried flow-through fractions were redissolved in 20 μl of 5% formic acid and applied to the C18 microcolumns. The columns were then washed with 20 μl of 5% formic acid, and subsequently peptides were eluted with 2 × 20 μl of 50% methanol and 2% formic acid. The eluates were dried and redissolved in 0.5% formic acid for LC-MS/MS. The nano-LC-MS/MS analysis was performed with a nanoACQUITY ultraperformance liquid chromatography (UPLC) system (Waters) connected to a hybrid linear ion trap/orbitrap tandem mass spectrometer (Thermo Electron). Dissolved peptides were loaded at 500 nl/min into a pulled and fused silica capillary with an inner diameter of 75 μm and a tip of 8 μm (New Objective) packed to a length of 12 cm with reversed-phase ReproSil-Pur C18-AQ 3-μm resin (Maisch) and eluted at 200 nl/min by a stepwise 180-min gradient of 0–100% buffer A (0.2% formic acid in water) and buffer B (0.2% formic acid in acetonitrile). The linear ion trap/orbitrap (LTQ-Orbitrap) mass spectrometer was operated in a data-dependent MS/MS mode. Survey full-scan MS spectra (from m/z 300 to 2000) were acquired in the orbitrap with a resolution of 60,000 at m/z 400. A maximum of five peptides were sequentially isolated for fragmentation in the linear ion trap using collision-induced dissociation (CID). The lock mass option was enabled to improve mass accuracy as described (27.Olsen 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 (1246) Google Scholar). All spectra were acquired with Xcalibur software. MS spectra were searched via MASCOT (28.Perkins D.N. Pappin D.J. Creasy D.M. Cottrell J.S. Probability-based protein identification by searching sequence databases using mass spectrometry data.Electrophoresis. 1999; 20: 3551-3567Crossref PubMed Scopus (6814) Google Scholar) (version 2.2.0, Matrix Science, London, UK). Searches were performed against the International Protein Index (IPI) human (version 3.48; 71,400 protein entries) database that was concatenated with decoy database sequences (142,800 protein entries in total) (29.Elias J.E. Gygi S.P. Target-decoy search strategy for increased confidence in large-scale protein identifications by mass spectrometry.Nat. Methods. 2007; 4: 207-214Crossref PubMed Scopus (2873) Google Scholar). Peak lists were generated using MaxQuant (30.Cox J. Mann M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification.Nat. Biotechnol. 2008; 26: 1367-1372Crossref PubMed Scopus (9467) Google Scholar) (version 1.0.12.5) with the following parameters: top six MS/MS peaks for 100 Da, three data points for centroid, Gaussian centroid determination, slice peaks at local minima. MaxQuant identifies potential SILAC pairs based on characteristics such as mass differences of labeled amino acids and intensity correlation over elution time to establish peptide peak lists. MaxQuant was also used for SILAC quantitation, false discovery rate (FDR) determination, peptide/protein grouping, phosphorylation site localization probability scoring, and data filtering based on MASCOT search results. The initial precursor mass tolerance was set to ±7 ppm, whereas an accuracy of ±0.5 Da was used for MS/MS fragmentation spectra. Enzyme specificity was set to trypsin, allowing cleavages N-terminal to proline. Carbamidomethylation was set as fixed modification. Oxidation, protein N-terminal acetylation, Arg10, Lys8, and phosphorylation (Ser/Thr/Tyr) were considered as variable modifications. A maximum of three labeled amino acids and two missed tryptic cleavages and a minimum peptide length of six amino acids were allowed. A minimum of one unique peptide was required for protein identification. In the case where the set of identified peptides in one protein was equal to or completely contained in the set of identified peptides of another protein, these two proteins were joined in the same protein group. Shared peptides are most parsimoniously associated within the group of the protein with the highest number of identified peptides but remain in all groups where they occur (30.Cox J. Mann M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification.Nat. Biotechnol. 2008; 26: 1367-1372Crossref PubMed Scopus (9467) Google Scholar). The protein with the highest number of identified peptides is presented as the leading protein in the supplemental tables with the highest confidence in its protein group. To ensure the fidelity of protein and phosphorylation site identifications, the target-decoy database strategy was used to minimize FDRs in our data set (29.Elias J.E. Gygi S.P. Target-decoy search strategy for increased confidence in large-scale protein identifications by mass spectrometry.Nat. Methods. 2007; 4: 207-214Crossref PubMed Scopus (2873) Google Scholar). A composite database is created by a “target” protein sequence database appropriate to the protein mixture to be analyzed (IPI human version 3.48), and a “decoy” database is created by reversing the target protein sequences. Because the reverse transformation preserves amino acid frequencies, protein and (approximate) peptide length distributions, and approximate mass distributions of theoretical peptides, the number of spectra matching decoy sequences gives an estimate of FDR. Estimated peptide/protein FDR was calculated as follows: (Number of hits in the reversed database/Number of hits in the forward database) × 100% (31.Choi H. Nesvizhskii A.I. False discovery rates and related statistical concepts in mass spectrometry-based proteomics.J. Proteome Res. 2008; 7: 47-50Crossref PubMed Scopus (169) Google Scholar). A maximum 5% peptide FDR and 2% protein FDR were allowed for the first step peptide/protein identification. A MASCOT ion score of at least 12 and phosphorylation site localization probability equal to or higher than 75% were used for data filtering. However, we included in a separate table (supplemental Table S5, A and B) all down-regulated phosphorylation sites (see below) with lower MASCOT scores (7 ≤ x < 12) identified from inhibitor (supplemental Table S5A) and shRNA (supplemental Table S5B) experiments as some of these sites might also be functionally relevant and may be considered for validation by in vitro spotting experiments (see below) despite their lower confidence. Automated quantitation was accomplished by the MaxQuant software. To correct for possible changes of protein levels on the mitotic spindle, the SILAC ratio of each phosphorylated peptide was normalized to the SILAC ratio of the corresponding protein (calculated as the mean of all unmodified peptide ratios from this protein). To compare the protein levels on isolated spindles with those in total lysates by MS (see Fig. 5A), ratios (H/L: TAL/MA) on the spindle were normalized against the average ratio of all α/β-Tubulin subunits identified to correct for variability in spindle isolation (supplemental Table S4A). Next, the ratios from the two spindle experiments (biological replicates) were averaged, and proteins with ratios that followed a different trend between biological replicates were excluded (above and below 1 ± 0.2). To define the relative change in spindle localization, the averaged normalized ratio for each protein from the spindle preparation experiment was divided by the corresponding normalized ratio from total lysates. The orbitrap raw data (MS raw files), MS/MS peak lists, and MS/MS spectra associated with this study were submitted to the Tranche data repository and can be downloaded from https://proteomecommons.org/tranche/ using the following hash and pass phrase: Santamariaetal: zt4vKJ7TYsdufcxOekHmhXJAmlNxac61TD8lMT/CWcE6LaOVcB/no0hnzd2ws3cZrvmApH65svbnb0QtkFuc0T85GtkAAAAAACJdhQ==. Motif-X was used with a significance cutoff of 0.0001 and at least 10 occurrences of the motif in the set. IPI human was used as a background data set with the built-in function. STRING 8.0 was queried with human protein symbols from supplemental Table S2, A and B. Confidence was set to medium (0.400), and only options “Databases” and “Experiments” were selected. Conservation of phosphorylated residues was checked with BLAST searches against rat (RGSC3.4.54), mouse (NCBIM37.54), zebrafish (Zv8.54), Drosophila melanogaster (BDGP5.4), Xenopus tropicalis (JGI4.1.54), Caenorhabditis elegans (WS190.54), and Saccharomyces cerevisiae (SGD1.0) proteomes obtained from the Ensembl FTP server. First, close sequence homologs of the proteins were identified using an E-value cutoff of 1e−50. Then, the phosphorylated site was checked for exact conservation in the alignment. Peptide arrays were constructed using standard Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry on a MultiPep robotic spotter according to the manufacturer's directions (Intavis). For Plk1 assays on peptide-immobilized cellulose membranes, dried membranes were first washed in ethanol and then hydrated in kinase buffer (50 mm Tris-HCl, pH7.5, 10 mm MgCl2, 1 mm DTT, 100 μm NaF, and 10 μm sodium orthovanadate) for 1 h followed by overnight blocking in kinase buffer with 100 mm NaCl and 0.5 mg/ml BSA. The next day, the membrane was blocked again with kinase buffer containing 1 mg/ml BSA, 100 mm NaCl, and 10 μm cold ATP at 30 °C for 45 min. The block was subsequently replaced with kinase reaction buffer containing 0.2 mg/ml BSA, 50 μCi/ml [γ-32P]ATP (3000 Ci/mmol, 10 mCi/ml), 150 nm recombinant His-Plk1, and 10 μm ATP for 3 h on a shaker at 30 °C. The membranes were then washed extensively (10 × 15 min in 1 m NaCl, 3 × 5 min in H2O, 3 × 15 min 5% H3PO4, and 3 × 5 min in H2O) and then sonicated overnight in 8 m urea, 1% SDS (w/v), and 0.5% (v/v) β-mercaptoethanol to remove residual nonspecific radioactivity. The membranes were washed again with H2O followed by ethanol and dried before being visualized by autoradiography. Aurora A assays were performed similarly but using Aurora A kinase buffer (20 mm Hepes, pH 7.4, 150 mm KCl, 5 mm MnCl2, 5 mm NaF, and 1 mm DTT). Sites were considered positive when differential signals could be observed between the Ser/Thr/Tyr and the Ala version of the peptide, negative when no detectable signal was observed, and non-conclusive when the peptide was also phosphorylated on the membrane incubated with ATP alone as control or alternatively when the signal between the Ser/Thr/Tyr and the Ala version of the peptide was indistinguishable. To investigate the Plk1-dependent phosphoproteome of human mitotic spindles, we used a quantitative phosphoproteomics strategy that combines SILAC with selective enrichment of spindle-associated proteins. To enable quantitation of the changes of phosphopeptide abundance by MS, cells were labeled by growing them in medium containing either normal arginine and lysine (Arg0/Lys0) or the heavy isotopic variants [13C6,15N4]arginine and [13C6,15N2]lysine (Arg10/Lys8) (20.Ong S.E. Blagoev B. Kratchmarova I. Kristensen D.B. Steen H. Pandey A. Mann M. Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics.Mol. Cell. Proteomics. 2002; 1: 376-386Abstract Full Text Full Text PDF PubMed Scopus (4625) Google Scholar) (Fig. 1A). To synchronize control cells at a similar mitotic stage and, importantly, to obtain similar microtubule arrays as in Plk1-inactivated cells, we interfered with the function of the kinesin motor protein Eg5. Like Plk1 inactivation (32.Lane H.A. Nigg E.A. Antibody microinjection reveals an essential role for human polo-like kinase 1 (Plk1) in the functional maturation of mitotic centrosomes.J. Cell Biol. 1996; 135: 1701-1713C" @default.
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• W2146453026 date "2011-01-01" @default.
• W2146453026 modified "2023-10-11" @default.
• W2146453026 title "The Plk1-dependent Phosphoproteome of the Early Mitotic Spindle" @default.
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• W2146453026 doi "https://doi.org/10.1074/mcp.m110.004457" @default.
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