Quantitative Phospho-proteomics toInvestigate the Polo-like Kinase 1-DependentPhospho-proteome*□S

Karin Grosstessner-Hain‡, Bjorn Hegemann‡¶¶, Maria Novatchkova‡,Jonathan Rameseder¶�, Brian A. Joughin�, Otto Hudecz§, Elisabeth Roitinger‡,Peter Pichler‡‡, Norbert Kraut§§, Michael B. Yaffe�**, Jan-Michael Peters‡��,and Karl Mechtler‡§��

Polo-like kinase 1 (PLK1) is a key regulator of mitoticprogression and cell division, and small molecule inhibi-tors of PLK1 are undergoing clinical trials to evaluate theirutility in cancer therapy. Despite this importance, currentknowledge about the identity of PLK1 substrates is lim-ited. Here we present the results of a proteome-wideanalysis of PLK1-regulated phosphorylation sites in mi-totic human cells. We compared phosphorylation sitesin HeLa cells that were or were not treated with thePLK1-inhibitor BI 4834, by labeling peptides via methylesterification, fractionation of peptides by strong cationexchange chromatography, and phosphopeptide en-richment via immobilized metal affinity chromatography.Analysis by quantitative mass spectrometry identified4070 unique mitotic phosphorylation sites on 2069 pro-teins. Of these, 401 proteins contained one or multiplephosphorylation sites whose abundance was decreasedby PLK1 inhibition. These include proteins implicated inPLK1-regulated processes such as DNA damage, mitoticspindle formation, spindle assembly checkpoint signaling,and chromosome segregation, but also numerous pro-teins that were not suspected to be regulated by PLK1.Analysis of amino acid sequence motifs among phosphor-ylation sites down-regulated under PLK1 inhibition in thisdata set identified two potential novel variants of the PLK1consensus motif. Molecular & Cellular Proteomics 10:10.1074/mcp.M111.008540, 1–11, 2011.

Progression through the cell cycle is controlled by cyclin-dependent kinases (CDKs)1. In mitosis, several other kinases,including Aurora A and B (AURKA/B) and PLK1, are activatedto orchestrate the different events that are required for chro-mosome segregation and subsequent cell division. PLK1 hasseveral essential roles during mitotic entry, early mitosis, andlate mitosis (1, 2). Before mitotic entry, PLK1 is required forthe release from a DNA-damage-induced G2-phase arrest (3).During mitotic entry, PLK1 amplifies cyclin-dependent kinase1 (CDK1) activation, enabling efficient onset of mitosis (4) andmediates centrosome maturation, the accumulation of �-tu-bulin complexes on centrosomes (5, 6). In prometaphase,PLK1 is required for the generation of stable kinetochore-microtubule attachments (7–10). PLK1 also promotes disso-ciation of cohesin from chromosome arms in prophase andprometaphase by phosphorylating cohesin’s STAG2 subunit(11–14), as well as multiple aspects of cytokinesis by phos-phorylating activators and effectors of RhoA (1, 15).

For each of these processes, only few PLK1 substrateshave been identified so far, and in most cases potential sub-strates have often only been identified by testing candidateproteins in in vitro kinase assays, lacking the context of cel-lular regulatory systems (16, 17). The function of PLK1 inthese processes is therefore incompletely understood. Fur-thermore, it remains to be determined if PLK1 also phosphor-ylates proteins that have functions in cellular processes otherthan the ones mentioned above. Because PLK1 is essentialfor cell division and because its inhibition leads to a mitoticarrest followed by apoptotic cell death (8) several small mol-ecule inhibitors of PLK1 are presently undergoing clinical trialsto test their potential utility in cancer therapy (reviewed in

From the ‡Research Institute of Molecular Pathology (IMP), 1030Vienna, Austria; §Institute of Molecular Biotechnology (IMBA), 1030Vienna, Austria; ¶Computational Systems Biology Initiative; �KochInstitute for Integrated Cancer Research; **Departments of Biologyand Biological Engineering, Massachusetts Institute of Technology,77 Massachusetts Ave Cambridge, MA 02139; ‡‡Christian DopplerLaboratory for Proteome Analysis, 1030 Vienna, Austria; and §§Boeh-ringer-Ingelheim, 1121 Vienna, Austria; ¶¶Present address: Instituteof Biochemistry, Eidgenossische Technische Hochschule (ETH)Zurich, CH-8093 Zurich, Switzerland

Received February 7, 2011, and in revised form, July 12, 2011Published, MCP Papers in Press, August 21, 2011, DOI

10.1074/mcp.M111.008540

1 The abbreviations used are: CDK, cyclin-dependent kinase;PLK1, Polo-like kinase 1; SCX, strong cation exchange chromatog-raphy; AURKA/B, Aurora A and B; FT, flow through; CENPF, centro-mere protein F; WAPAL, wings apart-like; EIF4B, eukaryotic transla-tion initiation factor 4B; noc, nocodazole; ph, phospho; I, BI 4834induced; S, BI 4834 sensitive; N-R, BI 4834 unregulated; MSA, mul-tistage activation collisional induced dissociation; IVK, in vitro kinaseassay.

Research© 2011 by The American Society for Biochemistry and Molecular Biology, Inc.This paper is available on line at http://www.mcponline.org

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18, 19). A more comprehensive knowledge about the identityof PLK1 substrates will therefore not only be important tounderstand the role of PLK1 in basic cellular functions, butalso to understand the cellular effects of PLK1 inhibitors incancer patients. We therefore developed a systematic, pro-teome-wide approach for the unbiased identification of po-tential PLK1 substrates by combining treatment of humanmitotic cultured cells with a highly selective PLK1 inhibitorwith quantitative mass spectrometric analysis of phospho-peptides. This approach led to the identification of 519 PLK1inhibitor sensitive phosphorylation sites on 401 proteins butalso revealed that the abundance of 134 phosphorylation siteson 122 proteins was increased upon inhibition of PLK1. Theseresults provide important new insight into the functions ofPLK1.

EXPERIMENTAL PROCEDURES

Cell Synchronization—The medium composition was used as de-scribed (20). For cell cycle synchronization HeLa cells were firstarrested at 50% confluency and a second time after release into freshmedium by using 2 mM thymidine (Sigma-Aldrich) followed by asecond release. Seven hours after the second release cells werearrested in prometaphase with 330 nM nocodazole (noc) for 3 h.Inhibition of PLK1 for the time course experiment was achieved using250 nM of BI 4834 during the last 15, 30, 45, 60, or 120 min of thenocodazole arrest. Prometaphase cells were harvested by a mitoticshake-off, washed twice with PBS (containing noc or noc and BI4834, respectively, in the same concentrations as in the cell culturebuffer), frozen in liquid nitrogen and stored at �80 °C.

Immunofluorescence Microscopy—After harvesting and washingwith PBS, cells were cytospun (Thermo Fisher Scientific, ShandonBrand) and fixed onto microscopy slides (12). The nuclear envelopesof the cells were stained with a Lamin A antibody and DNA wascounterstained with 4�-6-Diamidino-2-phenylindole (DAPI, MolecularProbes, Invitrogen, UK). For further details see Supplemen-tary experimental procedures.

Sample Preparation for MS Analysis—Cell lysis was performed asdescribed in Poser et al. (21). Proteins were precipitated in ice-coldacetone for further purification before proteolytic cleavage (seeSupplementary experimental procedures).

Sample preparation included enzymatic digestion with Lys-C andtrypsin and was followed by isotopic labeling. The two samples weredifferentially labeled with either heavy or light methanol and subse-quent desalting was performed as previously described (22). Driedpeptide pellets (each containing 2 mg of trypsin-digested cell lysate)were dissolved in 1.5 ml of strong cation exchange (SCX) buffer A(buffer composition and the SCX separation gradient are shown in theSupplementary experimental procedures). The peptides were sepa-rated on a PolySULFOETHYL A column (Poly LC, USA) of the dimen-sion: 4.6 mm ID � 15 cm length (operated at 1 ml/min) using a 100min separation gradient. Elution of peptides was facilitated by in-creasing pH and salt concentration in parallel. Twenty-five separatefractions were collected, desalted and phosphopeptides were en-riched by IMAC as described in Morandell et al. (22).

Nano-HPLC Separation—Chromatographic separation of enrichedphosphopeptides and unphosphorylated peptides from the IMACflow-through was performed on an UltiMate Plus Nano-LC chroma-tography system (consisting of Famos, Switchos, UltiMate pumpingsystem and UV detector, Dionex Benelux). First, samples were ap-plied to a reversed-phase trap column (PepMap C18, 5 mm � 0.3mm � 5 �m, 100 Å) and washed with 0.1% trifluoroacetic acid

(Pierce, Thermo Fisher Scientific) at a flow rate of 20 �l/min for 40 min.Separation of peptides was achieved on a C18 column (PepMap C18,150 mm � 0.075 mm � 3 �m, 100 Å) at a flowrate of 275 nl/min. Forall liquid chromatography (LC)-MS experiments, a linear gradient from100% solvent A (5% acetonitrile (Sigma-Aldrich), 0.1% formic acid(Merck, Germany)) to 70% solvent B (30% acetonitrile, 0.08% formicacid) in 205 min and further to 100% solvent B in 15 min was appliedfor peptide separation. This separation was followed by a high or-ganic wash with 90% solvent C (80% acetonitrile, 10% trifluoroetha-nol (Sigma-Aldrich), 0.08% formic acid) for the next 10 min.

MS detection—The nano-HPLC system was directly coupled to ahybrid linear ion trap/Fourier transform ion cyclotron resonance massspectrometer (LTQ-FT Ultra, Thermo Fisher Scientific) with a 7-Teslasuperconducting magnet (LTQ-FT Ultra was used for the definition ofthe cutoff) or an LTQ Orbitrap XL hybrid mass spectrometer (ThermoFisher Scientific) via a nano-electrospray ionization source (ProxeonBiosystems, Denmark). Metal-coated nano ESI emitters (New Objec-tive) generated the spray at a needle voltage of 2 kV. Mass spectrawere acquired in positive ionization mode with the following settings:Fullscan: 400–1800 Th., resolution 60,000, AGC target � 3e5 for LTQOrbitrap XL measurements, resolution 100,000, AGC target � 5e5 forLTQ-FT measurements. For all full scan measurements in the orbitrapdetector a lock-mass from siloxane (m/z 429.088735) was used forinternal calibration as described earlier (23). In the applied MSmethod, fragmentation was performed on the five most intense sig-nals of the survey scan using collisional induced dissociation withmultistage activation of the neutral loss of phosphoric acid at 49 and32.7 Da (24). Singly charged ions were excluded from precursorselection and precursors of MS2 spectra acquired in previous scanswere temporarily restricted for further fragmentation for a period of 3min with an exclusion mass window from –0.005 Da to � 2 Da aroundthe precursor m/z.

Data QualityLabeling Efficiency by Flow Through Measurement—The unphos-

phorylated peptides descending from the IMAC flow-through (flow-through data set contains pools 1–25 merged into one file for thedetermination of the labeling efficiency) was searched twice. Forpeptide identification of the flow through data, all MS/MS spectrawere searched using Mascot 2.2.0 (Matrix Science, London, UK). Thegeneration of dta-files for Mascot was performed using the ExtractMSn program (version 4.0, Thermo Fisher Scientific). First the lightesterification was applied as fixed modification and second, theheavy modification on the C-term and DE in addition to the followingmodifications: peptide mass tolerance � 3ppm; fragment mass tol-erance � 0.5 Da; database nr-human 2010–06-28 comprising232,368 sequences containing 82,489,472 residues, trypsin; fixedmodification carbamidomethyl (C); variable modifications oxidation(M), phosphorylation (STY); max. number of missed cleavages 3. Onepeptide was required for the protein identification. The two exportedpeptide lists were merged so that only the highest scoring peptideinterpretation for each spectrum was retained, and filtered to obtain alist of rank 1 peptides with an expect value � 0.05 and a minimumMascot score of 20. This resulted in 5940 peptide-spectrum matchescarrying one or more light methyl modifications, 6136 peptide-spec-trum matches carrying one or more heavy methyl modifications, and74 peptide-spectrum matches containing 76 DE residues that werenot methyl labeled at all (0.6%). Subsequently, the fraction of labeledglutamates, aspartates, and C termini was determined and the label-ing efficiency was calculated separately for the heavy and methylesterified samples, taking into account that completely unlabeledpeptides could be derived from either the heavy or the light methylesterification reactions.

Sequest Search Parameters for Phosphopeptide Identification,Cutoff Determination and False Discovery Rate Calculation—Acquired

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data (Xcalibur RAW-file) were converted into Sequest DTA-files usingBioworks Browser 3.3.1 (Thermo Fisher Scientific). DTA-files weresearched against the nr database (ftp://ftp.ncbi.nlm.nih.gov/blast/db/FASTA/nr.gz), version 2009–07-12 including only the human subsetof the database, named nr-human further on. This database wasgenerated using NCBI provided taxonomic information (453,136 se-quences; 163,150,522 residues) in combination with the reversedversion of the database generated with Bioworks (25, 26).

The modifications were as described above for determination ofthe regulatory cutoff, the peptide mass tolerance was set to �5 ppm.One peptide was required for the protein identification. To reach afalse discovery rate on the peptide and the protein level of 1%, filtercriteria were employed (see below).

Relative Quantification of Phosphopeptides—For quantification,the raw files derived from the phosphopeptide pools 1 to 25 and theinitial experiment to determine the cut-off were searched as de-scribed above, but only with the light methyl esterification as fixedmodification, because we could not detect a significant differencebetween the two (light/heavy) searches.

Results from database searches were then filtered for SEQUESTscores (XCorr of peptides (1�, 2�, 3�, 4�: 1.8, 2.2, 3.2, 4.0), Sf �0.45,peptide mass accuracy 5 ppm, percent ions �25) to contain onaverage 1% false-positive protein and peptide identification in all 25pools. The area of the heavier partner was integrated by the PepQuantool of the Bioworks software, applying a mass difference of 3.0188Da from the light precursor. All phosphopeptides were quantifiedusing the following quantification parameters: mass tolerance 0.05,minimum threshold 500, number of smoothing points 15. The calcu-lated regulatory ratios of all corresponding heavy and light peptidepairs were manually inspected and, if necessary, peak area integra-tion was corrected using PepQuan. Multiple protein isoforms wereindividually quantified, if the identified phosphopeptides clearly dis-tinguished the isoforms. Otherwise only the isoform with the highestnumber of peptides was presented in the data.

A list of all identified phosphopeptides with their correspondingscores is shown in supplementary Table S7.

Data Availability—All identified phosphopeptides together with theircorresponding spectra were uploaded to PRIDE (http://www.ebi.ac.uk/pride/) (27). The data can be downloaded from http://www.ebi.ac.uk/pride/ (accessions: 12092–12116 (pool1–25) and 13517–13519 (flow through data and cutoff experiment)).

Bioinfomatic Analysis—For every detected phosphopeptide,quantitative information obtained from the 25 SCX pools was com-bined to give a median ratio in case of multiple evidences; in caseof even number of observations the geometric mean of the medianpair was used. The normalization factor was determined by meas-uring the nonphosphopeptides (FT) of all 25 pools and calculatingthe median. The median log2 ratio of all nonphosphopeptides iden-tified was –0.03, therefore we did not consider it further.

Phosphopeptides with precise phosphorylation site localization bySEQUEST were considered separately, because of the higher confi-dence of the phospho-site to be dependent on PLK1 (sup-plementary Table S1). In case of doubly or triply phosphorylatedpeptides it was impossible to determine the PLK1-dependent phos-phorylation site (all phosphopeptides see supplementary Table S2).For the later motif and Scansite analysis, phosphopeptides containingonly one phosphorylation site, were generated as a 15-mer peptideconsisting of the site of phosphorylation and seven residues on boththe N- and C-terminal side.

Scansite analysis of all obtained phosphoproteins was performedas described in Obenauer et al. (28). For this analysis the wholeprotein sequence was searched for Scansite predictions. Later thephosphorylation sites obtained by the Scansite analysis with high andmedium thresholds were compared with the MS data. PSSMs for

CDK1, AURKA, AURKB, NEK2, and PLK1 were provided by the Yaffelaboratory (29). Two-sided Fisher’s exact test was performed to com-pare the incidence of kinase motifs in the BI 4834 sensitive, BI 4834induced and BI 4834 not-regulated sets.

A greedy search for statistically significantly enriched motifs in theup-regulated and down-regulated foregrounds (either BI 4834-sensi-tive or -induced), relative to the background of all sites identified inthis study, was carried out as described (30). Significances were notcalculated for, nor was search recursed on, any motif that was notcontained in at least 2% of the sites in the foreground being searched(see also supplementary Experimental Procedures).

RESULTS AND DISCUSSION

Identification of PLK1-dependent Phosphorylation Sites—Because PLK1 is active in mitosis we searched for phospho-peptides whose abundance is affected by inhibition of PLK1in cells arrested in prometaphase, using the strategy outlinedin Fig. 1A. We used the compound BI 4834 to inhibit PLK1 inthis study because this compound inhibits PLK1 with similarefficacy (IC50 7.6 nM) but with higher selectivity over therelated enzyme PLK3 (IC50 198.4 nM) as the previously char-acterized compound BI 2536 (8). Treatment of cells with in-creasing amounts of BI 4834 showed that at 250 nM BI 4834caused a cellular phenotype indistinguishable from BI 2536treatment, e.g. with prometaphase arrested cells displayingmonopolar spindles, reduced amounts of tubulin gamma 1(TUBG1) at centrosomes, and an abolished electrophoreticmobility shift of budding uninhibited by benzimidazoles 1B(BUB1B) and CDC25C (8)2.

To obtain a homogenous population of cells arrested inprometaphase we synchronized HeLa cells using a doublethymidine arrest and release protocol. Seven hours after thesecond release cells were arrested in prometaphase withnocodazole for 3 h. During this treatment, the PLK1 inhibitorBI 4834 was added for various periods of time to determinethe shortest inhibitor treatment necessary for full PLK1 inhi-bition and to keep secondary effects to a minimum. Mitoticcells were subsequently harvested by shake off (Fig. 1B).Immunofluorescence microscopy experiments revealed thatour synchronization protocol yielded a mitotic index of 94% inthe absence or presence of BI 4834 (supplementary Fig. S1).Synchronization of cells in G2/M-phase was also confirmedby FACS analysis (supplementary Fig. S1). Using 250 nM of BI4834 we tested five different treatment durations of 15, 30, 45,60, and 120 min. To measure PLK1 inhibition and subsequentdephosphorylation of PLK1 substrates we analyzed cell ly-sates by immunoblotting, using phospho-specific antibodiesthat recognize a PLK1-dependent phosphorylation site(S1224ph) on the cohesin subunit STAG2 (12, 31). This phos-phoepitope became undetectable after 15 min of BI 4834treatment (Fig. 1C). To address if BI 4834 also affected theactivities of other mitotic kinases we also analyzed phosphor-ylation of serine 10 on histone H3, a substrate of Aurora B (32),

2 Hegemann et al., paper in preparation.

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and the phosphorylation dependent electrophoretic mobilityshift of the APC/C subunit CDC27, which depends on CDK1activity (33). As an additional marker of CDK1 activity we alsoanalyzed threonine 14/tyrosine 15 phosphorylation on CDK1,modifications that are known to inhibit this enzyme. BI 4834treatment for 15 min did not cause a detectable reduction inH3 phosphorylation or of the CDC27 mobility shift or anincrease of inhibitory CDK1 phosphorylation, indicating thatthe phosphorylation states of Aurora B and CDK1 substrateswere not or not to the same degree affected as the phosphor-ylation of PLK1 substrates. These data are consistent with theprevious observation that inhibition of PLK1 causes activationof the spindle assembly checkpoint and an arrest in prometa-phase, which depends on Aurora B and CDK1 activity (8, 34).

Using this synchronization protocol we isolated HeLa cellsin prometaphase that were or were not treated for 15 min withBI 4834, digested proteins by trypsinization, and labeled pep-tides via methyl esterification with heavy and light methanolicHCl (35). The labeling efficiency was similar for both cell

populations (97.5% light label, 97.7% heavy label) as deter-mined by comparing all unphosphorylated peptides thatwere identified (supplementary Table S3). After the labelingstep the two peptide samples were combined, separated bySCX into 25 fractions, phosphopeptides were enriched fromeach fraction by Fe3�-IMAC and analyzed by massspectrometry.

We identified a total of 9313 phosphopeptides correspond-ing to 5742 unique phospho-peptides of which 1158 werephosphorylated on multiple sites and 4584 on a single site(singly phosphorylated peptides are listed in suppl-ementary Table S1, singly and multiply phosphorylated pep-tides are listed in supplementary Table S2). To unambiguouslyidentify PLK1-dependent phosphorylation sites we focusedour further analysis on the 4070 unique phosphorylation sitesidentified in the set of 4584 singly phosphorylated peptides.The 4070 phosphorylation sites analyzed were present on2069 proteins (supplementary Table S1) and 1262 of thesesites (31%) had not been reported before (www.phosphosite.

FIG. 1. Sample generation and control experiments. A, Flow scheme summarizing the experimental strategy to identify PLK1-inhibitor-sensitive phosphorylation sites (numbers in bold reflect singly and multiply phosphorylated peptides, all other numbers are based on singlyphosphorylated peptides). B, Time line to generate highly homogeneous mitotic cell cultures. Five different BI 4834 incubation times in the 3 hnocodazole arrest were tested. C, To identify the time of BI 4834 treatment required for full PLK1 inhibition with smallest indirect effect, sampleswere generated as described in B and analyzed for the mitotic status using phosphorylation of Histone H3 on serine 10 (H3 S10 ph) andphospho-dependent CDC27 shift, for CDK1 activity by probing CDK1 inhibitory phosphorylation on threonine 14 and tyrosine 15 (CDK1T14,Y15 ph) and for PLK1 activity by probing STAG2 phosphorylation on serine 1224 (STAG2 S1224 ph).

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org (36)). We considered phosphopeptides as being regu-lated by BI 4834 treatment if their abundance varied at leasttwofold because an analysis of identical mitotic samplesthat were differentially labeled with light and heavy metha-nol and analyzed as above, showed that 91% of all phos-phopeptides had less than twofold variation in abundancebetween the two samples (Fig. 2A, supplementaryFig. S2). Using this threshold we found 519 phosphorylationsites (12.8%) down-regulated whereas the abundance of134 phosphopeptides (3.3%) was increased (Table I,supplementary Table S1). At a regulatory cutoff above 1:45,no inhibitor-induced phosphorylation sites were detectedwhereas 51 inhibitor-sensitive sites were identified with aregulatory ratio ranging from 45:1 up to 86:1, the maximummedian ratio observed (supplementary Table S1). The 519down-regulated phosphorylation sites were present on 401proteins. These proteins may represent substrates of PLK1, orsubstrates of other protein kinases whose activity depends onPLK1, or in some cases substrates of unidentified kinases thatare also inhibited by BI 4834. Table II shows 32 proteins thatcontain three to nine BI 4834-sensitive phosphorylation sites.

In a biological replicate analysis we identified 6261 uniquephosphopeptides, confirming 42.5% of the 5742 uniquephosphopeptides initially identified. Of this set of commonpeptides, 60% confirmed the original regulatory category(peptides which were detected in both replicates withinthe same regulatory categories are listed in supple-mentary Tables S1 and S2).

The abundance of phosphopeptides might not only changedue to dephosphorylation, but also because of a change inprotein stability or because the monitored phosphopeptidebecomes phosphorylated at a site close to the one monitored.To test if protein stability is changing after 15 min BI 4834treatment we determined the abundance of 12523 nonphos-phorylated peptides, corresponding to 2761 proteins in the

samples obtained from cells treated with or without BI 4834.The median ratio of these peptides was 1:1.02, suggestingthat changes in protein levels had little if any influence on theabundance of phosphopeptides (Fig. 2A). To test if any of theidentified BI 4834-sentitive phosphorylation sites derive fromsingly phosphorylated phosphopeptides, which were phos-phorylated on a second site or if any of the BI 4834-inducedphosphorylation sites derive from doubly phosphorylatedpeptides on which one site was dephosphorylated, we iden-tified those singly phosphorylated peptides that overlappedwith multiply phosphorylated peptides. Because none of theBI 4834-sensitive phosphorylation sites and 4 (supple-mentary Table S4) of the identified BI 4834-induced phos-phorylation sites overlap with these 282 phosphopeptides,we can thus exclude that any of the changes in phospho-peptide abundance of BI 4834-sensitive phosphopeptideswe detected are attributable to additional phosphorylationof these peptides and that only in very few cases abun-dance of BI 4834-induced phosphopeptides are attributableto dephosphorylation of a residue on the same peptide.

To confirm some of the mass spectrometry data we raisedantibodies against BI 4834-sensitive phosphopeptides de-rived from centromere protein F (CENPF), wings apart-likeprotein (WAPAL) and the eukaryotic translation initiation factor4B (EIF4B). In immunoblot experiments, all three antibodiesrecognized bands at the expected size in extracts from mitoticHeLa cells, but not in extracts from cells synchronized inG2-phase, consistent with the possibility that these antibod-ies recognize only the mitotically phosphorylated forms oftheir antigens. Importantly, in all three cases the immunoblotsignals were reduced after 15 min of BI 4834 treatment, andthe extent of reduction correlated with the regulatory ratiosdetermined by quantitative mass spectrometry (Fig. 2B). Theresults indicate that the changes in phosphopeptide abun-dance detected by quantitative mass spectrometry are in

FIG. 2. Relative (phospho-)peptide frequency distribution and evaluation of MS data. A, Distribution of log2 ratios of all detectedphosphopeptides and nonphosphopeptides. In total 9313 phosphopeptides and 12635 unphosphorylated peptides were detected. B,Validation of PLK1 targets by Western blot analysis with phospho-specific antibodies against CENPF, WAPAL, and EIF4B. Bar diagramsindicate the corresponding ratios found by MS quantification.

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many if not all cases caused by changes in protein phosphor-ylation in the mitotic cells.

Before we carried out our analyses, 42 PLK1 proteins hadbeen reported to be PLK1 substrates, but for most of them theexact phosphorylation site had not been determined (1, 37).Our experiments identified phosphorylation sites in 29 of 42previously reported PLK1 substrates, but only seven ofthese contained BI 4834-sensitive phosphorylation sites,and only for CDC27 and KIF23 the same sites were foundregulated by us and the previous studies (Table III). Thissmall degree of overlap between our and previous resultscould be because of experimental differences becausemost previous studies determined phosphorylation sites byusing in vitro kinase reactions. However, we cannot excludethat our approach failed to detect some PLK1-regulatedphosphorylation sites, either because the correspondingphosphopeptides were not recovered, or because the 15min BI 4834 treatment was not sufficient to allow dephos-phorylation of phosphorylation sites that had been gener-ated by PLK1. Very recently, another phospho-proteomicstudy identified 866 PLK1-dependent phosphorylation siteson 304 mitotic spindle proteins (38). We identified only 25 ofthese 866 phosphorylation sites. Also in this case, the lowdegree of overlap might result from differences in experi-mental conditions, for example because Santamaria et al.used a different PLK1 inhibitor and shRNA-mediated PLK1depletion and cells already entered mitosis in the absenceof active PLK. However, it is also possible that both studiesonly identified subsets of the total number of PLK1-depend-ent phosphorylation sites in the human proteome.

Bioinformatic Analysis of BI 4834-regulated Phosphoryla-tion Sites—Using the PLK1 inhibitor we observed the reduc-tion of many phosphorylation sites but also the induction ofsome phosphorylation sites. Although we optimized for ashort inhibitor treatment, this observation suggests that weare also detecting secondary effects of PLK1 inhibition. Toassess which BI 4834-sensitive phosphorylation sites mightbe generated by PLK1 we made use of the Scansite algo-rithm. This algorithm identifies amino acid sequences matchingkinase consensus motifs based on in vitro kinase assays onoriented peptide libraries (28, 39). In addition to the initial set of28 kinase motifs (28) currently available in Scansite, the mitotickinases PLK1, Aurora kinase A and B, as well as NEK2 werealso recently characterized and included in Scansite (29). Weanalyzed our data set of 4070 phosphorylation sites for theexistence of all Scansite motifs and could relate 44% ofthe identified sites to 27 Scansite kinase motifs with mediumand high stringency. Interestingly, the 27 kinase motifs thatshowed significant hits in the total data set of phosphorylationsites are mostly associated with kinases implicated in mitosis(49% match to motifs for the major mitotic kinases AURKA/B,PLK1, and CDK1 (Scansite uses three distinct motifs for CDK1:Cdc2, CDK1.1, and CDK1.2) and NEK2 (Fig. 3A and sup-plementary Fig. S3). Importantly, the PLK1 motif is 4-fold en-riched among BI 4834-sensitive phosphorylation sites, whilenone of the other kinase motifs show significant enrichment inthe set of BI 4834-sensitive phosphorylation sites. This indicatesthat our approach identified 12 potential direct PLK1 substrates:NFKB2, TUBA1C, PDCD11, CAMSAP1L1, ZNF295, GOLGA2,NCK1, OPTN, NUMA1, INF2, RAD18, and COPS8. The analysis

TABLE INumber of unique peptides (phosphorylation sites in brackets) containing one or multiple phosphorylation sites with different cutoffs (1:2, 1:5,

and 1:10)

UniqueNumber of phosphopeptides with one or

more phosphorylation sites%

Number of phosphopeptides with onephosphorylation sites

%

Total number ofphosphopeptides

5742 (5643) 100 4584 (4070) 100

BI 4834-sensitivephosphopeptides(cutoff 1:2)

713 (761) 12 559 (519) 12

BI 4834-inducedphosphopeptides(cutoff 2:1)

157 (175) 3 135 (134) 3

BI 4834-sensitivephosphopeptides(cutoff 1:5)

379 (412) 7 293 (274) 6

BI 4834-inducedphosphopeptides(cutoff 5:1)

17 (21) �1 14 (14) �1

BI 4834-sensitivephosphopeptides(cutoff 1:10)

267 (292) 5 211 (200) 5

BI 4834-inducedphosphopeptides(cutoff 10:1)

6 (8) �1 5 (5) �1

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of all phosphopeptides with 2 or more phosphorylation sitesrevealed similar results (supplementary Fig. S4). Besides thesignificant distribution of the PLK1 motif in this analysis also theCK2 motif hits are significantly distributed. The increase inmatches to the CK2 motif in the BI 4834 induced pool mightsuggest a repressing function of PLK1 activity toward CK2substrate phosphorylation.

About 60% of the inhibitor-sensitive sites cannot bematched to the PLK1 motif or any of the other Scansitemotifs. This suggests that either PLK1 or other kinases canphosphorylate sites that differ from their characterized con-sensus motif or that other, yet unidentified kinases gener-ated these sites. Similarly, many of the inhibitor-inducedphosphorylation sites could not be matched to any of the 32kinase motifs.

To identify amino sequence motifs that are enrichedamong the inhibitor-sensitive or inhibitor-induced phosphor-ylation sites, we used a previously described algorithm (30).All 134 BI 4834-induced and 519 BI 4834-sensitive phos-phorylation sites were transformed into 15-mer peptidesconsisting of the site of phosphorylation and seven residueson both the N- and C-terminal sides (shown insupplementary Table S1). 15-mers from the induced andsensitive data subsets were analyzed for motifs enriched incomparison to the entire set of phosphorylation sites. Twomotifs predicted to be highly surface accessible were sig-nificantly enriched in the induced phosphopeptide group byeight- and 19-fold, respectively (Fig. 3B, logos a and b (40),supplementary Table S4). The motifs generally show a ba-sophilic group on the positions upstream of the phospho-

TABLE IIProteins with at least three BI 4834-sensitive phosphorylation sites

Entrez-GeneID Entrez name Protein-nameBI 4834-sensitivephospho-peptides

Implicated in

3875 KRT18 keratin 18 9 Member of the intermediate filament genefamily with KRT8

3856 KRT8 keratin 8 9 Cellular structural integrity10579 TACC2 transforming, acidic coiled-coil

containing protein 26 Centrosome- and microtubule-interacting

protein9414 TJP2 tight junction protein 2 5 Proper assembly of tight junctions4008 LMO7 LIM domain 7 5 Protein-protein interactions23451 SF3B1 splicing factor 3b, subunit 1,

155kDa4 Member of the U2 small nuclear

ribonucleoprotein complex1063 CENPF centromere protein F, 350/400ka

(mitosin)4 Chromosome segregation during mitotis

7916 BAT2 HLA-B associated transcript 2 4 Inflammatory process of pancreatic beta-cell destruction during the developmentof insulin-dependent diabetes mellitus

7165 TPD52L2 tumor protein D52-like 2 3 Uncharacterized254427 C10orf47 chromosome 10 orf 47 3 Uncharacterized7175 TPR translocated promoter region (to

activated MET oncogene)3 Directly interacts with several components

of the nuclear pore complexes22837 COBLL1 COBL-like 1 3 Uncharacterized6294 SAFB scaffold attachment factor B 3 Attaching the base of chromatin loops to

the nuclear matrix8661 EIF3A eukaryotic translation initiation

factor 3, subunit A3 Development and differentiation

26135 SERBP1 SERPINE1 mRNA bindingprotein 1

3 Tumor invasion and metastasis

85379 CTA-221G9.4 KIAA1671 protein 3 Uncharacterized4791 NFKB2 nuclear factor of kappa light

polypeptide gene enhancer inB-cells 2 (p49/p100)

3 Apoptosis, inappropriate immune celldevelopment, delayed cell growth

85456 TNKS1BP1 tankyrase 1 binding protein 1 3 Assembly of bipolar spindles23271 CAMSAP1L1 calmodulin regulated spectrin-

associated protein 1-like 13 Uncharacterized

9100 USP10 ubiquitin specific peptidase 10 3 Cleavage of ubiquitin from ubiquitin-conjugated substrates

55604 LRRC16A leucine rich repeat containing 16A 3 Uncharacterized283489 ZNF828 zinc finger protein 828 3 Uncharacterized10397 NDRG1 N-myc downstream regulated 1 3 Stress responses, hormone responses,

cell growth, differentiation andapoptosis

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rylation sites and acidophilic residues downstream of thissite. These motifs, which match on 9 and 6 of 134 sites,respectively, point to kinases (or phosphatases), which aredirectly or indirectly inhibited by PLK1. In the BI 4834-sensitive pool five significantly overrepresented motifs werefound (Fig. 3, logos c–g). The first three motifs (c–e), sur-rounding either serine alone (c: 48 peptide-matches of 518)or serine and threonine (d: 51 peptide-matches of 518),have aspartic acid at position –2, or in the case of motif (e),aspartic or glutamic acid in this position (78 peptide-matches of 518). These three motifs are on average 1.7- to2.4-fold enriched within the BI 4834-sensitive sites andtogether cover 34% of all detected PLK1 inhibitor sensitivephosphorylation sites (supplementary Table S5). These re-sults suggest that PLK1 might not strictly require the hydro-phobic residue in the �1 position as was observed in sev-eral PLK1 substrates (2, 41). Two more motifs (Fig. 3, f–g)include an asparagine at position –2 and either a hydropho-bic amino acid on position �1 (in the case of phospho-serine or -threonine, 12 peptide-matches of 518) or a hy-drophilic residue at position �3 (for phospho-serine alone,14 peptide-matches of 518). The enrichment for those mo-tifs was 3.4 fold and 3.8 fold respectively. We detected aPLK1-dependent phosphorylation site matching motif 3f in aseparate study on S153 of the RAD21 protein3. By generating aphospho-specific antibody against RAD21 S153 ph we couldconfirm that this site is PLK1-dependent (supplemen-tary Fig. S5). Recently, asparagine in the –2 position was alsodetected by a peptide library-based approach to find PLK1motifs (42) and in a phospho-proteomic approach by Santama-ria et al. (38) supporting the hypothesis this motif represent anew variant of the PLK1 consensus site.

Our bioinformatic analysis thus identified two potentialvariations of the published PLK1 consensus motif (43). Bothrevealed an asparagine at position –2 and in motif (f) alsothe leucine at –3 is prominent. In a recent summary of PLK1substrates (2), nine out of 40 substrates contained a D/E at–2 without a hydrophobic residue in position �1 whereas sixout of 40 substrates contained an asparagine in position –2.

Our results suggest that these motif variations are quitecommon in PLK1 substrates. These variations should thusbe taken into account when searching for PLK1 substratesby sequence analysis. 22 other motifs were found whichcontain a tryptophan in close proximity to the phosphory-lation site. One reason for this environmental tryptophan-enrichment could be the preference of the PLK1 inhibitortoward tryptophan-containing phosphorylation sites, butfurther experiments have to be done to test this assumption(supplementary Table S6).

To classify the identified BI 4834-regulated phosphopro-teins, we allocated a subset of them into five biological pro-cesses associated with mitosis (Fig. 3C) derived from manualliterature curation in combination with GO term analysis (44).This places the identified substrates into the PLK1-regulatedprocesses DNA damage, mitotic progression regulation, mi-totic spindle formation, chromosome segregation and spindleassembly checkpoint signaling.

Until recently, PLK1’s only role in DNA damage was thoughtto be the promotion of degradation of the mitotic entry inhibitorsWEE1 and Claspin to release cells from the G2 damage check-point. A recent publication (3), however, identified TP53BP1, amediator protein important early in the DNA damage response,and Chk2 as PLK1 targets required for DNA damage checkpointrelease. In our set of PLK1 targets we found a large number ofproteins involved in DNA damage detection and repair (Fig. 3C).These findings thus support the idea that efficient inactivation ofthe DNA damage checkpoint requires phosphorylation and pos-sibly inactivation of a large set of DNA damage response com-ponents. The cohesin-complex holds sister chromatids to-gether from S-phase to mitosis (45). Its dissociation fromchromosome arms in prophase depends on WAPAL and onphosphorylation of STAG2 by PLK1 (12, 31). Here, we alsoidentified the cohesin-associated proteins PDS5A/B, and WA-PAL as potential in vivo PLK1 targets. This finding will enablefurther analysis of the role of PLK1 in cohesin release fromchromosomes. Furthermore, PLK1 is required for proper kine-tochore function to allow faithful chromosome segregation andis also enriched at kinetochores during mitosis (2). Nevertheless,only few PLK1-substrates at the kinetochore are known to date.We identified the published substrate BUB1 for which the PLK13 Hegemann et al., in preparation.

TABLE IIIComparison of PLK1 substrates reported in the literature with proteins containing BI 4834 sensitive phosphorylation sites. (* Phosphorylation

sites that were reported in the literature and found to be BI 4834-sensitive (IVK in vitro kinase assay, MS: mass spectrometry))

ProteinBI 4834-sensitive

phosphosite(s)ID method reported phosphosite(s) Ref.

BUB1 T306 IVK ND Qi W et al., 2006CDC27 S427* IVK, MS T209, S427*, S434, S435 Kraft et al., 2003KIF23 S911*, S912*, T913 IVK S911*, S912* Liu et al., 2004ASPM S348, S367, S370 IVK ND Maria do Carmo Avides et al., 2001TB53BP1 S1618, T591, S598, S692 IVK ND van Vugt et al., 2010CDC25B S563 ND Valerie Lobjois et al., 2009PIN1 S41 IVK S65 Eckerdt et al., 2005

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phosphorylation site, however, has not been described before(46, 47). In addition we found the kinetochore regulatory proteinZWINT (48) and the mitotic checkpoint activator MAD1L1 (49) tocontain BI 4834-sensitive phosphorylation sites. Interestingly,also several components of the NUP107–160 nuclear pore sub-complex were detected as potential PLK1 substrates; consist-

ent with the possibility that nuclear pore disassembly in mitosisis regulated by PLK1.

CONCLUSIONS

By using quantitative phosphoproteomics in combinationwith a small molecule kinase inhibitor we have identified a

FIG. 3. Bioinformatic analysis of the PLK1-dependent phospho-proteome and functional annotation of PLK1-dependent pro-teins. A, Extracted phosphorylation site-centered 15-mers of all unique phosphorylation sites were compared with Scansite predictionsfor 27 kinases (only five are shown, see supplementary Fig. S3 for full analysis). Only top-scoring kinase hits were accepted for each15-mer to avoid redundancy. Predicted PLK1 targets are significantly enriched in the BI 4834-sensitive data set compared with thenonsensitive, which is indicated by the Fisher’s Exact test p value of 3.905E-08 (*). B, Kinase consensus logos significantly enriched ineither of the BI 4834-regulated data sets. The enrichment of the logo in comparison to the not-regulated (N-R) phosphopeptides is shownas bar diagram (S for sensitive, I for induced). Selected logos from the BI 4834-sensitive phosphopeptides (c-e) resemble the reported PLK1consensus sequence whereas logos f and g represent a newly discovered potential PLK1 motif. C, Selected phospho-proteins showing BI4834-dependent regulation according to subcellular localization and biological function are listed according to their regulatory ratio.

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large number of potential PLK1 substrates. This method rep-resents a generic method applicable for the identification ofother kinase substrates in vivo for which inhibitors are avail-able. Of 4070 identified mitotic phosphorylation sites, motifanalysis assigned 46% of the sites to 27 kinases and half of allsites were matched to mitotic kinases. Within the 519 BI4834-sensitive phosphorylation sites, the PLK1 motif wassignificantly enriched, indicating that some if not many ofthese sites are direct targets of PLK1. Further, unbiased motifanalysis identified a minimal PLK1 motif containing only D/E inposition –2 and a variation of the classical PLK1-motif con-taining N at position –2. Only few of the known PLK1 sub-strates contain the novel motif N-x-S/T or D/E-x-S/T respec-tively. We have now identified many PLK1 substrates withthese motifs, suggesting D/E-x-S/T and N-x-S/T represent anew subclass of PLK1 motifs. Our data provide an importantresource for further biological and biochemical experimentsinvestigating the function of PLK1. Further, as PLK1 is over-expressed in many cancers and PLK1 inhibitors are in clinicaltrials as anti-cancer drugs, the identified PLK1 substrates mayalso serve as powerful diagnostic or clinical biomarkers.

Acknowledgments—We thank M. Mazanek, M. Madalinski, G.Krssakova, I. Steinmacher, J. R. Hutchins, R. Imre, A. Schmidt, Jo-hann Holzmann, Peter Lenart and Thomas Kocher for their excellentassistance.

* This work was funded by the APP within the Austrian GenomeProgram (GEN-AU), by the European Commission via the SixthFramework Programme Integrated Project MitoCheck (LSHG-CT-2004-503464), from NCI grant U54-CA112967, NIEHS grantES015339 and an International Fulbright Science and TechnologyAward. Work in the laboratories of J.-M. P. and K. M. receivedsupport from Boehringer Ingelheim.

□S This article contains supplemental material.�� To whom correspondence should be addressed: Jan-Michael

Peters, Research Institute of Molecular Pathology, Dr. Bohrgasse 7,1030 Vienna. Tel.: �43 1 79730-3250; Fax: �43 1 798 71 53; E-mail:peters@imp.ac.at or Karl Mechtler, address as J-M P., Tel.: �43 179730-4280; Fax: �43 1 798 71 53; E-mail: mechtler@imp.ac.at.

REFERENCES

1. Petronczki, M., Lenart, P., and Peters, J. M. (2008) Polo on the Rise-fromMitotic Entry to Cytokinesis with Plk1. Dev. Cell. 14, 646–659

2. Park, J. E., Soung, N. K., Johmura, Y., Kang, Y. H., Liao, C., Lee, K. H.,Park, C. H., Nicklaus, M. C., and Lee, K. S. (2010) Polo-box domain: aversatile mediator of polo-like kinase function. Cell Mol. Life. Sci. 67,1957–1970

3. van Vugt, M. A., Gardino, A. K., Linding, R., Ostheimer, G. J., Reinhardt,H. C., Ong, S. E., Tan, C. S., Miao, H., Keezer, S. M., Li, J., Pawson, T.,Lewis, T. A., Carr, S. A., Smerdon, S. J., Brummelkamp, T. R., and Yaffe,M. B. (2010). A mitotic phosphorylation feedback network connectsCdk1, Plk1, 53BP1, and Chk2 to inactivate the G(2)/M DNA damagecheckpoint. PLoS. Biol. 8, e1000287

4. O’Farrell, P. H. (2001) Triggering the all-or-nothing switch into mitosis.Trends. Cell Biol. 11, 512–519

5. Lane, H. A., and Nigg, E. A. (1996) Antibody microinjection reveals anessential role for human polo-like kinase 1 (Plk1) in the functional mat-uration of mitotic centrosomes. J. Cell Biol. 135, 1701–1713

6. Sunkel, C. E., and Glover, D. M. (1988). polo, a mitotic mutant of Drosophiladisplaying abnormal spindle poles. J. Cell Sci. 89, 25–38

7. 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., and Lee, K. S. (2006)Self-regulated Plk1 recruitment to kinetochores by the Plk1-PBIP1 inter-action is critical for proper chromosome segregation. Mol. Cell. 24,409–422

8. Lenart, P., Petronczki, M., Steegmaier, M., Di Fiore, B., Lipp, J. J., Hoff-mann, M., Rettig, W. J., Kraut, N., and Peters, J. M. (2007) The small-molecule inhibitor BI 2536 reveals novel insights into mitotic roles ofpolo-like kinase 1. Curr. Biol. 17, 304–315

9. Sumara, I., Gimenez-Abian, J. F., Gerlich, D., Hirota, T., Kraft, C., de laTorre, C., Ellenberg, J., and Peters, J. M. (2004) Roles of polo-like kinase1 in the assembly of functional mitotic spindles. Curr. Biol. 14,1712–1722

10. van Vugt, M. A., van de Weerdt, B. C., Vader, G., Janssen, H., Calafat, J.,Klompmaker, R., Wolthuis, R. M., and Medema, R. H. (2004) Polo-likekinase-1 is required for bipolar spindle formation but is dispensable foranaphase promoting complex/Cdc20 activation and initiation of cytoki-nesis. J. Biol. Chem. 279, 36841–36854

11. Gimenez-Abian, J. F., Sumara, I., Hirota, T., Hauf, S., Gerlich, D., de laTorre, C., Ellenberg, J., and Peters, J. M. (2004) Regulation of sisterchromatid cohesion between chromosome arms. Curr. Biol. 14,1187–1193

12. Hauf, S., Roitinger, E., Koch, B., Dittrich, C. M., Mechtler, K., and Peters,J. M. (2005) Dissociation of cohesin from chromosome arms and loss ofarm cohesion during early mitosis depends on phosphorylation of SA2.PLoS. Biol. 3, e69

13. Losada, A., Hirano, M., and Hirano, T. (2002) Cohesin release is required forsister chromatid resolution, but not for condensin-mediated compaction,at the onset of mitosis. Genes. Dev. 16, 3004–3016

14. Sumara, I., Vorlaufer, E., Stukenberg, P. T., Kelm, O., Redemann, N., Nigg,E. A., and Peters, J. M. (2002) The dissociation of cohesin from chromo-somes in prophase is regulated by Polo-like kinase. Mol. Cell. 9, 515–525

15. Burkard, M. E., Maciejowski, J., Rodriguez-Bravo, V., Repka, M., Lowery,D. M., Clauser, K. R., Zhang, C., Shokat, K. M., Carr, S. A., Yaffe, M. B.,Clauser, K. R., Zhang, C., Shokat, K. M., Carr, S. A., Yaffe, M. B., andJallepalli, P. V. (2009). Plk1 self-organization and priming phosphoryla-tion of HsCYK-4 at the spindle midzone regulate the onset of division inhuman cells. PLoS. Biol. 7, e1000111

16. Snead, J. L., Sullivan, M., Lowery, D. M., Cohen, M. S., Zhang, C., Randle,D. H., Taunton, J., Yaffe, M. B., Morgan, D. O., and Shokat, K. M. (2007)A coupled chemical-genetic and bioinformatic approach to Polo-likekinase pathway exploration. Chem. Biol. 14, 1261–1272

17. Yun, S. M., Moulaei, T., Lim, D., Bang, J. K., Park, J. E., Shenoy, S. R., Liu,F., Kang, Y. H., Liao, C., Soung, N. K., et al. (2009) Structural andfunctional analyses of minimal phosphopeptides targeting the polo-boxdomain of polo-like kinase 1. Nat. Struct. Mol. Biol. 16, 876–882

18. Strebhardt, K., and Ullrich, A. (2006) Targeting polo-like kinase 1 for cancertherapy. Nat. Rev. Cancer. 6, 321–330

19. Taylor, S., and Peters, J. M. (2008) Polo and Aurora kinases: lessonsderived from chemical biology. Curr. Opin. Cell Biol. 20, 77–84

20. Hutchins, J. R., Toyoda, Y., Hegemann, B., Poser, I., Heriche, J. K., Sykora,M. M., Augsburg, M., Hudecz, O., Buschhorn, B. A., Bulkescher, J.,Conrad, C., Comartin, D., Schleiffer, A., Sarov, M., Pozniakovsky, A.,Slabicki, M. M., Schloissnig, S., Steinmacher, I., Leuschner, M., Ssykor,A., Lawo, S., Pelletier, L., Stark, H., Nasmyth, K., Ellenberg, J., Durbin, R.,Buchholz, F., Mechtler, K., Hyman, A. A., and Peters, J. M. (2010)Systematic analysis of human protein complexes identifies chromosomesegregation proteins. Science. 328, 593–599

21. Poser, I., Sarov, M., Hutchins, J. R., Heriche, J. K., Toyoda, Y., Pozniak-ovsky, A., Weigl, D., Nitzsche, A., Hegemann, B., Bird, A. W., Pelletier, L.,Kittler, R., Hua, S., Naumann, R., Augsburg, M., Sykora, M. M.,Hofemeister, H., Zhang, Y., Nasmyth, K., White, K. P., Dietzel, S., Mech-tler, K., Durbin, R., Stewart, A. F., Peters, J. M., Buchholz, F., and Hyman,A. A. (2008) BAC TransgeneOmics: a high-throughput method for explo-ration of protein function in mammals. Nat. Methods. 5, 409–415

22. Morandell, S., Grosstessner-Hain, K., Roitinger, E., Hudecz, O., Lindhorst,T., Teis, D., Wrulich, O. A., Mazanek, M., Taus, T., Ueberall, F., Mechtler,K., and Huber, L. A. (2010) QIKS - Quantitative identification of kinasesubstrates. Proteomics. 10, 2015–2025

23. Olsen, J. V., de Godoy, L. M., Li, G., Macek, B., Mortensen, P., Pesch, R.,Makarov, A., Lange, O., Horning, S., and Mann, M. (2005) Parts permillion mass accuracy on an Orbitrap mass spectrometer via lock mass

Polo-like Kinase 1-Dependent Phospho-proteome

10.1074/mcp.M111.008540–10 Molecular & Cellular Proteomics 10.11

injection into a C-trap. Mol. Cell Proteomics. 4, 2010–202124. Schroeder, M. J., Shabanowitz, J., Schwartz, J. C., Hunt, D. F., and Coon,

J. J. (2004) A neutral loss activation method for improved phosphopep-tide sequence analysis by quadrupole ion trap mass spectrometry. Anal.Chem. 76, 3590–3598

25. Elias, J. E., Haas, W., Faherty, B. K., and Gygi, S. P. (2005) Comparativeevaluation of mass spectrometry platforms used in large-scale proteom-ics investigations. Nat. Methods. 2, 667–675

26. Olsen, J. V., Blagoev, B., Gnad, F., Macek, B., Kumar, C., Mortensen, P.,and Mann, M. (2006) Global, in vivo, and site-specific phosphorylationdynamics in signaling networks. Cell. 127, 635–648

27. Vizcaino, J. A., Cote, R., Reisinger, F., Foster, J. M., Mueller, M.,Rameseder, J., Hermjakob, H., and Martens, L. (2009) A guide to theProteomics Identifications Database proteomics data repository. Pro-teomics. 9, 4276–4283

28. Obenauer, J. C., Cantley, L. C., and Yaffe, M. B. (2003) Scansite 2.0:Proteome-wide prediction of cell signaling interactions using short se-quence motifs. Nucleic. Acids. Res. 31, 3635–3641

29. Alexander, J., Lim, D., Joughin, B. A., Hegemann, B., Hutchins, J. R.,Ehrenberger, T., Ivins, F., Sessa, F., Hudecz, O., Nigg, E. A., Musacchio,A., Stukenberg, P. T., Mechtler, K., Peters, J. M., Smerdon, S. J., andYaffe, M. B. Spatial exclusivity combined with positive and negativeselection of phosphorylation motifs is the basis for context-dependentmitotic signaling. Sci. Signal. 4, ra42

30. Joughin, B. A., Naegle, K. M., Huang, P. H., Yaffe, M. B., Lauffenburger,D. A., and White, F. M. (2009) An integrated comparative phosphopro-teomic and bioinformatic approach reveals a novel class of MPM-2motifs upregulated in EGFRvIII-expressing glioblastoma cells. Mol. Bio-syst. 5, 59–67

31. Kueng, S., Hegemann, B., Peters, B. H., Lipp, J. J., Schleiffer, A., Mechtler,K., and Peters, J. M. (2006) Wapl controls the dynamic association ofcohesin with chromatin. Cell. 127, 955–967

32. Hsu, J. Y., Sun, Z. W., Li, X., Reuben, M., Tatchell, K., Bishop, D. K.,Grushcow, J. M., Brame, C. J., Caldwell, J. A., Hunt, D. F., Lin, R., Smith,M. M., Allis, C. D. (2000) Mitotic phosphorylation of histone H3 is gov-erned by Ipl1/aurora kinase and Glc7/PP1 phosphatase in budding yeastand nematodes. Cell. 102, 279–291

33. Kraft, C., Herzog, F., Gieffers, C., Mechtler, K., Hagting, A., Pines, J., andPeters, J. M. (2003) Mitotic regulation of the human anaphase-promotingcomplex by phosphorylation. EMBO. J. 22, 6598–6609

34. Petronczki, M., Glotzer, M., Kraut, N., and Peters, J. M. (2007) Polo-likekinase 1 triggers the initiation of cytokinesis in human cells by promotingrecruitment of the RhoGEF Ect2 to the central spindle. Dev. Cell. 12,713–725

35. Platt, M. D., Salicioni, A. M., Hunt, D. F., and Visconti, P. E. (2009) Use ofdifferential isotopic labeling and mass spectrometry to analyze capaci-tation-associated changes in the phosphorylation status of mouse spermproteins. J. Proteome. Res. 8, 1431–1440

36. Hornbeck, P. V., Chabra, I., Kornhauser, J. M., Skrzypek, E., and Zhang, B.(2004) PhosphoSite: A bioinformatics resource dedicated to physiologi-cal protein phosphorylation. Proteomics. 4, 1551–1561

37. Li, J., Wang, J., Jiao, H., Liao, J., and Xu, X. Cytokinesis and cancer: Pololoves ROCK‘n’ Rho(A). J. Genet. Genomics. 37, 159–172

38. Santamaria, A., Wang, B., Elowe, S., Malik, R., Zhang, F., Bauer, M.,Schmidt, A., Sillje, H. H., Koerner, R., and Nigg, E. A. (2010). ThePlk1-dependent phosphoproteome of the early mitotic spindle. Mol. CellProteomics 10, M110 004457

39. Yaffe, M. B., Leparc, G. G., Lai, J., Obata, T., Volinia, S., and Cantley, L. C.(2001) A motif-based profile scanning approach for genome-wide pre-diction of signaling pathways. Nat. Biotechnol. 19, 348–353

40. Crooks, G. E., Hon, G., Chandonia, J. M., and Brenner, S. E. (2004)WebLogo: a sequence logo generator. Genome Res. 14, 1188–1190

41. Lowery, D. M., Lim, D., and Yaffe, M. B. (2005) Structure and function ofPolo-like kinases. Oncogene. 24, 248–259

42. Jes Alexander, D. L., Joughin, B. A., Hegemann, B., Hutchins, J. R. A.,Ehrenberger, T., Ivins, F., Sessa, F., Hudecz, O., Nigg, E., Fry, A. M.,Musacchio, A., Stukenberg, P. T., Mechtler, K., Peters, J., Smerdon,S. J., and Yaffe M. B. (2011). Spatial exclusivity combined with positiveand negative motif selection for phosphorylation is the babsis for con-text-dependent mitotic signaling. Sci. Signal. 4, ra42

43. Nakajima, H., Toyoshima-Morimoto, F., Taniguchi, E., and Nishida, E.(2003) Identification of a consensus motif for Plk (Polo-like kinase) phos-phorylation reveals Myt1 as a Plk1 substrate. J. Biol. Chem. 278,25277–25280

44. Ashburner, M., Ball, C. A., Blake, J. A., Botstein, D., Butler, H., Cherry, J. M.,Davis, A. P., Dolinski, K., Dwight, S. S., Eppig, J. T., Harris, M. A., Hill,D. P., Issel-Tarver, L., Kasarskis, A., Lewis, S., Matese, J. C., Richardson,J. E., Ringwald, M., Rubin, G. M., Sherlock, G. (2000) Gene ontology: toolfor the unification of biology. The Gene Ontology Consortium. Nat.Genet. 25, 25–29

45. Peters, J. M., Tedeschi, A., and Schmitz, J. (2008) The cohesin complexand its roles in chromosome biology. Genes. Dev. 22, 3089–3114

46. Baumann, C., Korner, R., Hofmann, K., and Nigg, E. A. (2007) PICH, acentromere-associated SNF2 family ATPase, is regulated by Plk1 andrequired for the spindle checkpoint. Cell. 128, 101–114

47. Qi, W., Tang, Z., and Yu, H. (2006) Phosphorylation- and polo-box-depend-ent binding of Plk1 to Bub1 is required for the kinetochore localization ofPlk1. Mol. Biol. Cell 17, 3705–3716

48. Cheeseman, I. M., and Desai, A. (2005) A combined approach for thelocalization and tandem affinity purification of protein complexes frommetazoans. Sci. STKE. 2005, pl1

49. Sironi, L., Mapelli, M., Knapp, S., De Antoni, A., Jeang, K. T., and Musac-chio, A. (2002) Crystal structure of the tetrameric Mad1-Mad2 corecomplex: implications of a ‘safety belt’ binding mechanism for the spin-dle checkpoint. EMBO. J. 21, 2496–2506

Polo-like Kinase 1-Dependent Phospho-proteome

Molecular & Cellular Proteomics 10.11 10.1074/mcp.M111.008540–11