Quantitative phosphoproteomics reveals new roles for the ... · PP6-regulatedphosphorylationevents.Furthermore,wecharacterizedarole for PP6 in chromosome condensation byopposing casein

R E S E A R C H R E S O U R C E

C E L L C Y C L E

Quantitative phosphoproteomics reveals new rolesfor the protein phosphatase PP6 in mitotic cellsScott F. Rusin,1 Kate A. Schlosser,2 Mark E. Adamo,2 Arminja N. Kettenbach1,2*

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Protein phosphorylation is an important regulatory mechanism controlling mitotic progression. Proteinphosphatase 6 (PP6) is an essential enzymewith conserved roles in chromosome segregation and spin-dle assembly from yeast to humans. We applied a baculovirus-mediated gene silencing approach to de-plete HeLa cells of the catalytic subunit of PP6 (PP6c) and analyzed changes in the phosphoproteomeand proteome in mitotic cells by quantitative mass spectrometry–based proteomics. We identified 408phosphopeptides on 272 proteins that increased and 298 phosphopeptides on 220 proteins that de-creased in phosphorylation upon PP6c depletion in mitotic cells. Motif analysis of the phosphorylatedsites combinedwith bioinformatics pathway analysis revealed previously unknownPP6c-dependent reg-ulatory pathways. Biochemical assays demonstrated that PP6c opposed casein kinase 2–dependent phos-phorylation of the condensin I subunit NCAP-G, and cellular analysis showed that depletion of PP6cresulted in defects in chromosome condensation and segregation in anaphase, consistent with dysregu-lation of condensin I function in the absence of PP6 activity.

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INTRODUCTION

During mitosis, a cell undergoes dramatic changes in cellular structureand organization to divide its cytoplasm and organelles and equally seg-regate its genome to generate two viable daughter cells. Dynamic andreversible protein phosphorylation mediated by protein kinases andphosphatases is an important regulatory mechanism in this process (1, 2).Errors in cell division can lead to cells with aberrant chromosome num-ber, a state known as aneuploidy, which is a hallmark of human cancerand the origin of many birth defects. The family of phosphoprotein phos-phatases (PPPs), which includes PP1, PP2A, PP4, PP5, PP6, and PP7, is re-sponsible for the turnover of most serine and threonine phosphorylationevents in mitosis (1–3). Marine toxins, such as okadaic acid, calyculin A,and microcystin-LR, inhibit multiple members of the PPP family. Indeed,exposure of interphase cells to these compounds induces a pseudomitoticstate characterized by chromosome condensation and Golgi fragmenta-tion, and their application to mitotic cells inhibits exit from mitosis (4, 5).Thus, tight regulation of PPP activities is essential for accurate mitoticprogression.

Protein phosphatase 6 (PP6) is an essential trimeric holoenzyme thatconsists of one catalytic (PP6c), one of three regulatory (PPP6R1, PPP6R2,and PPP6R3), and one of three scaffolding [ankyrin repeat domain-containing protein 28 (ANR28), ANR44, and ANR52, respectively] sub-units (6–8). This combinatorial modularity results in nine distinct PP6holoenzymes and is likely responsible for the diverse regulatory functionsof PP6 by influencing localization, substrate targeting, and catalytic ac-tivity (9–20). Furthermore, PP6 regulatory subunits mediate interactionsbetween the holoenzyme and protein kinases, such as DNA-dependentprotein kinase (DNA-PK) (14), Aurora kinase A (AURKA) (21), trans-forming growth factor b–activated kinase (TAK1) (9), and kinases in theHippo pathway (16). PP6 is highly conserved in eukaryotes from yeastto humans and has a conserved role in chromosome segregation and spin-dle assembly inmitosis. In fissionyeast, the PP6c homolog Ppe1 promoteschromosome segregation (22), whereas in budding yeast, the PP6c homo-log Sit4 is required for G1 to S progression (23, 24). In Caenorhabditis

1Department of Biochemistry, Geisel School of Medicine at Dartmouth, Hanover,NH 03755, USA. 2Norris Cotton Cancer Center, Geisel School of Medicine atDartmouth, Lebanon, NH 03756, USA.*Corresponding author. E-mail: [email protected]

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elegans, PP6 regulates spindle positioning (25). In mitotic human cancercells, PP6 inhibits the activity of the mitotic kinase AURKA by dephos-phorylating Thr288 in the activation T-loop (21). Furthermore, PP6 opposesthemitotic kinase Polo-like kinase 1 (PLK1) by dephosphorylating a PLK1-dependent phosphorylation site on DNA-PK (20).

Genome-wide exome sequencing of malignant melanomas has iden-tified somatic mutations in the gene encoding PP6c (26, 27). Biochem-ical characterization found that these mutations are heterogeneous withregards to their effect on PP6c activity, stability, and subunit binding. Con-sequently, themutant enzymes can function as loss-of-function tumor sup-pressors or gain-of-function oncoproteins (17, 18, 28). In glioblastoma,the abundance of PP6c is increased and negatively correlated with patientsurvival (29). Although reduced PP6 activity might contribute to cancerinitiation or progression through activation of AURKA and promotion ofthe cell cycle, increased PP6 activity can promote DNA damage repairthrough activation of DNA-PK (12, 14, 30). Additional evidence for theimportance of PP6 in cancer comes from a mouse model of skin carcino-genesis in which deletion of PP6c promotes tumor formation upon expo-sure of the skin to a tumor initiator (31).

The role of PP6 is complex, and PP6 integrates multiple pathways es-sential for cell cycle progression. To determine PP6c-dependent biologicalprocesses and pathways inmitotic cells, we analyzed phosphorylation andprotein abundance changes upon PP6c depletion in Taxol-arrested HeLacells. Because of the lack of specific PP6 inhibitors and biochemical strate-gies, such as substrate-trappingmutants that can be used for tyrosine-directedprotein phosphatases (32, 33), we combined baculovirus-mediated RNA in-terference andmass spectrometry–based quantitative proteomics to identifyPP6-regulated phosphorylation events. Furthermore,we characterized a rolefor PP6 in chromosome condensation by opposing casein kinase 2 (CK2)–mediated phosphorylation of the condensin I subunit G (NCAP-G).

RESULTS

Generation of a BacMam short hairpin RNA strategy forPP6c depletionTo determine changes in protein phosphorylation upon PP6c depletion inmitotic cells, we used a baculovirus-based short hairpin RNA (shRNA)

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strategy. PP6c is encoded by an essential gene, and long-term reduction ofPP6c abundance results in mitotic defects (21, 31). Thus, constitutive de-pletion or deletion of the gene encoding PP6c in human cells is not fea-sible. To overcome the limitations of transient small interfering RNA(siRNA) transfection in large-scale phosphoproteomic experiments,which require ~1 × 107HeLa cells per condition and per replicate, we usedan shRNA-encoding virus. We generated a recombinant baculovirus–mediated shRNA expression system because recombinant baculovirusesare safe, stable for long-term storage, amenable to amplification, and caninfect awide range ofmammalian cells at high efficiency (34, 35). Recom-binant baculoviruses cannot replicate in organisms other than their insect

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cell host. To generate a recombinant bacu-lovirus expressing shRNAs, we combinedfeatures of baculoviral and lentiviral expres-sion vectors (see Materials and Methods fordetails) into a pFastBacMam-shRNA-GFPvector (Fig. 1A),which enabled virus gener-ation in Spodoptera frugiperda (Sf9) insectcells and shRNA and green fluorescent pro-tein (GFP) expression in mammalian cells.

To identify sequences for efficient PP6cdepletion, we tested five shRNA sequencestargeting the PP6c coding sequence (seeMaterials and Methods for shRNA se-quences). To determine the efficiency of in-fection, we infected HeLa cells with controlshRNA–encoding virus or virus encodingone of the five PP6c shRNAs andmonitoredGFP production after 48 hours.We observedgreater than 90% infection efficiency asdetermined by fluorescence microscopy ofGFP-positive cells (Fig. 1B and fig S1A).Two baculoviruses (PP6c-sh1 and PP6c-sh4)partially reduced PP6c abundance whenHeLa cells were infected with them individ-ually (fig. S1, B and C).When used in com-bination, viral infection with the same titerof virus but containing 50% encoding PP6c-sh1 and 50% encoding PP6c-sh4 produceda similar amount of GFP as infection witha 100% viral titer of either of the shRNA-encoding viruses (fig. S1, D and E). Moreimportantly, PP6c-sh1 combined with PP6c-sh4 reduced PP6c abundance to less than8%of that in cells infectedwith virus encod-ing a control shRNA (fig.S1, D and E).

To determinewhether the partial (~25%)reduction of PP6c abundance produced bythese individual shRNAs was sufficient tomodulate PP6c substrate phosphorylationin mitosis, we assessed the phosphorylationstatus of the PP6c-regulated Thr288 of theAURKAT-loop (21) byWestern blot analy-sis of infected HeLa cells that had beenarrested in mitosis (fig. S1, D and E). Thesedata indicated that the reduction in PP6 ac-tivity achieved with the individual shRNAswas insufficient to increase phosphoryl-ation at this PP6-regulated site but showedthat co-infection with both shRNA-encoding

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viruses produced a significant increase in phosphorylation of Thr288 ofAURKA in mitotic cells.

Off-target effects are a potential concern when using shRNA (36, 37).We reasoned that potential off-target effects are likely unique to individ-ual shRNAs and that the two shRNAswe used were unlikely to affect thesame off-target protein. Therefore, we compared protein abundance inmitotically arrested HeLa cells that had been infected with either controlshRNA–, PP6c-sh1–, or PP6c-sh4–encoding virus using multiplex quan-titative proteomics in biological triplicate to validate the on-target effectsof the shRNAs for PP6c (fig. S2A).We collected and digested total cellularprotein, labeled the peptides using tandem mass tag (TMT) reagents, and

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Fig. 1. Strategy to determine PP6c-dependent phosphorylation changes in cells by quantitative phospho-
proteomics. (A) Vector map of pFastBacMam-shRNA-GFP. SV40 PA term, simian virus 40 polyadenylationterminator; EGFP, enhancedGFP; CMV, cytomegalovirus promoter; Amp, ampicillin resistance gene.(B) Representative immunofluorescence micrographs of HeLa cells infected with dual PP6c shRNA- andGFP-expressing baculoviruses. Scale bar, 50 mm. GFP protein abundance was comparable for all testedPP6c shRNA and control viruses (fig. S1A). GFP protein abundance was performed in all experiments todetermine infection efficiency. DAPI, 4′,6-diamidino-2-phenylindole. (C) Western blot analysis of PP6cand GFP abundance in HeLa cells infected with viruses encoding control or both PP6c-1 and PP6c-4shRNAs (PP6c shRNA). Lamin A/C is the loading control. (D) Scheme depicting experimental strategy fordetermining PP6c-dependent changes in phosphorylation site occupancy upon PP6c depletion.MitoticallyarrestedHeLa cells infectedwith viruses encoding control or PP6c shRNAswere separately lysed, reduced,alkylated, and trypsin digested. Phosphopeptides (p-peptides) were enriched using titanium dioxide micro-spheres, heavy or light labeled by reductive dimethylation, andmixed. Phosphopeptideswere separated bystrong cation exchange (SCX) chromatography and analyzed by LC-MS/MS (n = 3 independentexperiments). (E) Western blot analysis of PP6c abundance and AURKA pThr288 in HeLa cell lysates asdescribed in (D). Lamin A/C is the loading control. (F) Quantification of PP6c, AURKA pThr288, and laminA/C abundance. PP6c and AURKA pThr288 abundances were normalized to Lamin A/C. *P < 0.05, ***P <0.001 (n = 3 independent experiments).





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analyzed the peptides by MS3-based quantitative liquid chromatography–tandem mass spectrometry (LC-MS/MS) (38). We identified and quanti-fied 6700 proteins with at least two unique peptides at a 1.2% protein-levelfalse discovery rate (FDR) in this analysis (fig. S2B and table S1).

Correlation analysis of the fold changes in protein abundance in cellsinfected with either of the two hairpins produced a correlation coefficientof R2 = 0.83 for proteins increased or decreased by 1.4-fold (log2 ratio<0.5 or > 0.5) (fig. S2B). Reassuringly, we observed the largest reductionin protein abundance in PP6c itself (fig S2B). Furthermore, we found thatthe abundance of the PP6c regulatory subunits PPP6R1, PPP6R2, andPPP6R3were also reduced, which is consistent with previous reports show-ing that siRNA-mediated reduction in the PP2A catalytic subunit resultedin a corresponding reduction in abundance of regulatory subunits (39),indicating that PPP regulatory subunit stability depends on the ability toform holoenzyme complexes. We ascribe the differences in the amount ofreduction of PPP6R1, PPP6R2, and PPP6R3 abundance to their differentabsolute protein abundance inHeLa cells (40), such that the relative higheramount of PPP6R3 resulted in greater destabilization upon depletion ofthe catalytic subunit of PP6 compared with that observed for the otherPPP6R subunits. Of the 6700 quantified proteins, only 35 exhibited sig-nificantly (P < 0.05, two-tailed Student’s t test) different amounts in cellsinfected with either of the two shRNA-encoding viruses (indicated by reddots in fig. S2B; table S1), withmost of these differences occurring at modestfold changes [median fold change of the most reduced protein ratio betweencells infected with each of the two hairpins: −0.08 (log2)], indicating thatthese are unlikely to be biologically important. On the basis of these analysesof the efficiency of PP6c depletion, the effect on AURKAThr288 phospho-rylation, and off-target effects, we combined viruses encoding each of thesetwo PP6c shRNAs for use in subsequent studies to ensure sufficient reduc-tion in PP6c abundance (Fig. 1C).

Quantitative phosphoproteomics of PP6c-depleted,mitotically arrested HeLa cellsAlthough PP6c plays an essential role in chromosome segregation andspindle assembly from yeast to humans (21, 22, 25), only two substratesof PP6c, Thr288 of AURKA and Ser3205 of DNA-PK, have been identifiedin mitotic cells (20, 21, 28). To identify additional PP6c-dependent path-ways important in mitosis, we infected HeLa cells with control shRNA–expressing virus or viruses expressing PP6c-sh1 and PP6c-sh4 andsynchronized the cells in mitosis using a thymidine block followed by re-lease into the microtubule-stabilizing drug Taxol (fig. S3). We collectedthe infected, mitotically arrested HeLa cells separately, lysed the cells, re-duced and alkylated the proteins, and digested the proteins into peptides(Fig. 1D). At this point, we took aliquots of the digested peptides for quan-tification of changes in protein abundance between the samples (not shownin Fig. 1D). We subjected the remaining peptides to phosphopeptide en-richment, chemical labeling for quantitative comparison, and LC-MS/MSanalysis. Both phosphopeptide and total peptide analyses were done inbiological triplicate.

We performedWestern blot analysis of PP6c abundance, aswell as thephosphorylation status of the known PP6c substrate Thr288 on AURKA,to confirm efficient depletion of PP6c (Fig. 1E). Quantification of Westernblot results normalized to the loading control lamin A/C showed that, uponinfection with viruses encoding PP6c-sh1 and PP6c-sh4, but not virusencoding control shRNA,PP6c protein abundancewas significantly reducedto barely detectable amounts, and that phosphorylation of Thr288 onAURKAwas significantly increased (Fig. 1F).

Overall, we identified and quantified 29,933 phosphopeptides on 4415proteins, of which 23,910 phosphopeptides were found in two of thethree biological replicates and 16,442 were found in all three (Fig. 2A

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and table S2). Becausewe reduced PP6c abundance for a prolonged period,we corrected for changes in protein abundance as well. Using the aliquotstaken after the peptide digestion, we quantified protein abundance from thethree biological replicates and corrected the phosphopeptide ratios on aper-replicate basis, which reduced the total number of phosphopeptidesquantified to 23,910 on 2931 proteins, of which 11,801 phosphopeptideson 2006 proteins were quantified in each of the three biological replicates(Fig. 2B).

We filtered the number of phosphopeptides and proteins to those thatincreased twofold or more in the PP6c-depleted cells with a P value of<0.1, which resulted in 658 phosphopeptides on 371 proteins (Fig. 2C).We chose this cutoff because phosphorylation site occupancy in mitoticcells is increased compared to that in interphase cells (41), and we rea-soned that additional increases upon PP6c depletion might be small. Ofthese 658 phosphopeptides, 408 were increased because of changes inthe phosphorylation abundance of the quantified phosphorylation site(fig S4A). These 408 phosphopeptidesmapped to 272 proteins, of whichmost (203) contained only a single phosphopeptide with a significantlychanged ratio (fig. S4B). Indeed, 71% of these proteins contained one ormore additional phosphorylation sites that did not change in abundance.That many of the phosphorylated sites that significantly changed were

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Ratio increase >twofold, P value <0.05, P value <0.1 Ratio decrease >twofold, P value <0.05, P value <0.1

Fig. 2. Candidate phosphorylation site substrates of PP6 in mitosis. (A)Venn diagrams depicting overlap of identified and quantified phospho-
peptides and proteins in biological triplicate experiments. (B) Venn di-agrams depicting overlap of identified and quantified phosphopeptidesand proteins after protein correction in these experiments. In (A) and (B),each experiment is represented by a color, and gray is the overlap in allthreeexperiments. (C) Volcanoplot of log2 ratios of protein-correctedphos-phopeptides from (B) plotted against the negative log10 of the P value oftheir fold change. We used the 298 phosphopeptides that decreasedand the 408 phosphopeptides that increased because of phosphoryl-ation abundance changes for subsequent analysis.





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on proteins that had other sites that did not change provided further supportthat this subset of the data represents phosphorylation site-specific changesand not differential protein abundance. As a positive control, one of the 408significantly increased phosphopeptides contained Ser3205 on DNA-PK(table S2 and fig. S5,A andB). The remaining 250 siteswere due to proteinabundance changes because the change in phosphopeptide abundancewas less than the change in protein abundance (table S2 and fig S4A).Furthermore, we also identified 519 phosphopeptides on 319 proteins thatwere decreased by twofold or more with a P value of <0.1, of which 298were due to changes in phosphopeptide abundance on 220 proteins and 221in protein abundance (table S2 and fig S4A). In the subsequent analyses ofPP6c-dependent signaling pathways, we concentrated on the 408 and 298phosphopeptides that increased or decreased, respectively, because of phos-phorylation changes upon PP6c depletion.

Chemical nature of PP6c-dependentphosphorylation sitesTo predict which kinases PP6c might oppose, we performed motif anal-ysis (42, 43) on the 408 or 298 phosphorylation sites that increased ordecreased upon PP6c depletion, respectively. In the phosphorylationsites that increased upon depletion of PP6c, we found an enrichmentof proline-directed (phospho-Ser/Thr-Pro), basophilic (Arg-X-phospho-Ser),and acidophilic (phospho-Ser-X-X-Glu) motifs (Fig. 3A), whereas the phos-phorylation sites that decreased upon depletion of PP6c were enriched forproline-directed (phospho-Ser/Thr-Pro) and basophilic (Arg-X-X-phospho-Ser) but not acidophilic motifs (Fig. 3B).

Because of the known role of PP6 in reducing AURKA activity (21),a kinasewith substrate preference for arginine-richmotifs (44), we expectedthe enrichment of a basophilic motif in phosphopeptides that exhibited anincrease in PP6-depleted cells. Furthermore, we considered that we couldassess the regulation ofAURKAsubstrates by comparing this PP6-dependentdata set with our previously published data set of AURKA substrates inTaxol-arrested HeLa cells that were identified using the AURKA-specificsmall molecule inhibitor MLN8054 (45) and quantitative phosphoproteo-mics (44). Of the 36 AURKA substrates present in the protein-correcteddata set from the PP6c-depleted cells (table S3), only 12were significantlyincreased in phosphorylation occupancy upon PP6c depletion (Fig. 3C).We also assessed what fraction of PP6c-dependent phosphorylation sitesthat contain the Arg-X-phospho-Ser motif were identified as AURKAsubstrates in theMLN8054 study (table S4). Of these 42 phosphopeptideswith the basophilic motif that increased in PP6c-depleted cells, 8 were re-ported as sensitive to AURKA inhibition, 18 were not significantly affectedby AURKA inhibition, and 15 were not identified or quantified in theMLN8054 study (Fig. 3D). Together, these results suggested that increasedAURKAactivity as indicated by increased phosphorylation of the AURKAactivation T-loop upon PP6c depletion results in increased phosphorylationsite occupancy of a subset of AURKA substrates.

In addition, the observed enrichment of proline-directed motifs wasalso not surprising because these are canonical kinase motifs for cyclin-dependent kinases (CDKs), including Cdk1, which functions in chromo-some condensation, nuclear envelope breakdown, organelle fragmentation,spindle assembly, and chromosome segregation (46–49). Proline-directedmotifs are also recognized by mitogen-activated kinases (MAPKs) (50)and glycogen synthase kinase 3 (GSK-3) (50). The acidophilicmotif couldbe ascribed to CK2, which requires an acidic residue at the third positiondownstream of the phosphorylatable amino acid (50–52).

Protein network analysisTo elucidate the biological processes regulated by PP6c, we performednetwork analysis on the 272 proteins that contain phosphorylation sites

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that increased upon PP6c depletion. We gathered protein-protein inter-action data among the 272 proteins from the STRING (Search Tool forthe Retrieval of Interacting Genes/Proteins) database (53) and analyzedthem in Cytoscape (54, 55) (fig. S6A). We determined densely connectedclusters of protein interactions within the network to identify nodes pre-dicted as involved in specific regulatory processes, which identified fourclusters with a P value of <0.01 (Fig. 4A). Gene ontology (GO) analysis ofeach of the four clusters indicated their enrichment in biological processesand cellular components (56). The cluster shown in black contained pro-teins implicated in chromosome segregation and condensation, with spin-dle and chromosomes as primary cellular components, which is consistentwith the conserved role of PP6 in chromosome segregation and spindleformation through the direct regulation of spindle and kinetochore pro-teins, including AURKA (21, 22, 25). However, the identification of pro-teins involved in chromosome condensation, including NCAP-G, SMC4,TOP2A, and KIF4A, as potentially regulated by PP6c is new, representinga previously unknown function of PP6c. The cluster shown in blue containedproteins involved in RNA processing that belong to nucleolar and preribo-somal complexes. During mitosis, the nucleolus is not present as a discretestructure, nucleolar proteins localize around chromosomes, and ribosomal

A Bp-Peptide ratio increase >2-fold

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Fig. 3. Motif analysis and comparison of candidate PP6c substrates with
known AURKA phosphorylation sites. (A) Enriched phosphorylation sitemotifs of phosphopeptides significantly increased by twofold ormore uponPP6c depletion. (B) Enriched phosphorylation site motifs of phosphopep-tides significantly decreased by twofold or more upon PP6c depletion. (C)Scatter plot of phosphopeptides that were previously ascribed to AURKAactivity versus their fold change upon PP6c depletion. (D) Scatter plot ofphosphopeptides that contain a basophilic [RXp(S/T)] motif and increaseby twofold or more upon PP6c depletion versus their fold change upon in-hibitionwithAURKA inhibitor. In (C) and (D), redcircles represent phospho-rylation sites regulated upon both AURKA inhibition and PP6c depletion,and black circles represent sites regulated upon either AURKA inhibition(C) or PP6c depletion (D). Dotted black lines indicate zero coordinates.



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DNA (rDNA) transcription is suppressed(57). Suppression of rDNA transcriptionand ribosomal RNA (rRNA) processing ismediated by protein phosphorylation inprophase by Cdk1 and, later in the cell cy-cle, is relieved by dephosphorylation (58).We also identified proteins involved in RNAsplicing and processing that are compo-nents of the spliceosome (yellow cluster).PP6c coimmunoprecipitates with small nu-clear ribonucleoproteins (snRNPs) that arepart of the spliceosome (59) and associateswith the spliceosome throughout the splic-ing reaction by binding to U1 snRNP (60).Protein phosphorylation is required for theformation of the spliceosome, and dephos-phorylation is necessary for progression ofthe catalytic splicing reaction (61). Phos-phatases might function in the exchange ofsnRNPs, regulate protein-protein interac-tions during splicing, or control splicing-independent functions of U1 snRNP (60).The fourth cluster of densely connected pro-teins contained proteins involved in proteintranslation on cytoplasmic ribosomes (ma-genta cluster). Like rDNA transcription andrRNA processing, most protein translationis suppressed in mitosis by protein phos-phorylation (62). PP6c activity might beinvolved in the reactivation of these pro-cesses in telophasewhennet phosphorylationby mitotic kinases decreases, and phospha-tase activity on these substrates can lead toa decrease in the phosphorylation of inhibito-ry sites in the protein translationalmachinery.

Performing the same type of analysison proteins that contain phosphorylationsites that decreased upon PP6c depletionresulted in a network with an overall lesswell connected structure (compare the num-ber of nodes that were not significantlydensely clustered in fig. S6, A and B). Weidentified three clusters of moderate con-nectivity (Fig. 4B), all of which had fewernodes than the clusters found in the inter-action network of proteins with increasedphosphorylation. We identified four proteinsinvolved in the regulation of Rab guanosinetriphosphatase activity (yellow cluster), aswell as a cluster (magenta) containing com-ponents of the nuclear envelope and nucle-ar core complex that regulateRNA transport.The largest cluster (shown in black) containsmicrotubule-associated proteins, such as thekinesinsKIF4A,KIF20, andKIF18B, whichare proteins that regulate microtubule-basedprocesses at the spindle (63, 64).

Forty proteins identified in our analysescontained phosphorylation sites that in-creased uponPP6c depletion,whereas other

MAPRE3

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Fig. 4. Processes regulated by PP6c in mitotic cells. (A) Subnetwork depicting the connectivity of highlyconnectedclusters in theprotein-protein interaction network fromphosphopeptides significantly increasedin phosphorylation upon PP6c depletion in mitotically arrested cells. Proteins in the different colored clus-ters and their enrichment in biological processes and cellular components are shown. (B) Subnetworkdepicting the connectivity of highly connected clusters in the protein-protein interaction network fromphosphopeptides significantly decreased in phosphorylation upon PP6c depletion inmitotically arrestedcells. Proteins in the different colored clusters and their enrichment in biological processes and cellularcomponents are shown.

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sites decreased in abundance (table S2). Someof these differentially occupied sites are re-lated. For instance,we observed an increasein phosphorylation of the doubly phospho-rylated peptide Ser4384:Ser4396 on Plectin,whereas the single phosphorylated peptideSer4384 decreased, which is suggestive ofstepwise enzyme activity. However, mostanticorrelated abundance changes of phos-phorylation sites on one protein appearedunrelated, suggesting more complex regula-tory mechanisms by PP6 holoenzymes andopposing kinases governing these proteinsin mitotic cells.

Investigation of PP6c- andCK2-regulated phosphorylationsite occupancy in NCAP-Gby in vitro assaysBy depleting PP6c for an extended period,we cannot distinguish the direct effect ofthe lack of PP6c phosphatase activity (asubstrate of PP6) from an indirect effectof PP6c depletion and reduced activity.Therefore, we conducted in vitro phospha-tase assays with purified PP6 holoenzymecomplex to determine whether PP6 directlydephosphorylated select phosphorylationsites from our data set. Because of the un-known role for PP6 in chromosome con-densation and the importance of properchromosome condensation in chromosomestability, we focused on the phosphorylationsite with increased abundance in PP6c-depleted mitotically arrested HeLa cellsidentified on NCAP-G, a member of thecondensin I complex.

We found that the abundance of a sin-gly phosphorylated peptide on NCAP-G(identified as either pSer973 or pSer975;hereafter referred to as pSer973/5) was sig-nificantly increased upon PP6c depletion(table S2 and fig. S7, A and B). We couldnot establish the specific site of phospho-rylation because the MS/MS spectra lackedthe site-determining ions required to distin-guish between them (fig. S7B). Thus, PP6cdepletion may affect either site or both ofthem when singly phosphorylated. Addi-tionally, we also observed the correspond-ing doubly phosphorylated (both pSer973

andpSer975; hereafter referred to as pSer973:975)peptide, which did not significantly changeupon PP6c depletion (table S2). To validatethat PP6c can directly dephosphorylateNCAP-G, we purified the condensin I com-plex from 293T cells stably expressing3×Flag-NCAP-H and purified the PP6 holo-enzyme from 293T cells stably expressing3×Flag-PP6c for an in vitro phosphatase

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Fig. 5. In vitro PP6 dephosphorylation analysis of NCAP-G. (A) Scheme depicting the experimental strategy for
PP6 invitrophosphataseassay.Purifiedcondensin Iwas incubatedwithorwithoutpurifiedPP6holoenzymeandresolvedbySDS-PAGE.NCAP-Gwasexcisedanddigested, andpeptidesweredifferentially labeledby reduc-tive dimethylation, mixed, and analyzed by LC-MS/MS (n = 3 independent experiments). (B) SDS-PAGE gel ofcondensin I purification. (C) Relative intensities of LC-MS/MS traces of heavy- and light-labeled phosphopep-tides corresponding to the singly phosphorylatedSer973/5 phosphorylation site inNCAP-G, extracted to±2partspermillion (ppm). Phosphopeptidesequence, charge state, and ionmass-to-charge ratios are indicated.Acidicamino acids are highlighted in red. (D) Relative intensities of LC-MS/MS traces of the heavy- and light-labeledunphosphorylated peptide spanning Ser973/5, extracted to ±2 ppm. Peptide sequence, charge state, and ionmass-to-charge ratiosare indicated.Acidicaminoacidsarehighlighted in red.Blue lines in (C)and (D) representthe samples incubated with buffer, and gray lines represent the samples incubated with purified PP6c.(E) Scheme depicting experimental strategy for CK2 in vitro kinase assay. Purified and l-phosphatase–dephosphorylatedcondensin Iwas incubatedwith orwithout purifiedCK2and resolvedbySDS-PAGE.NCAP-Gwas excised and digested, and peptides were labeled by reductive dimethylation, mixed, and analyzed byLC-MS/MS (n= 3 independent experiments). (F) Relative intensities of LC-MS/MS traces of heavy- and light-labeledphosphopeptidescovering theSer973/5phosphorylationsite inNCAP-G,extracted to±2ppm.Phospho-peptide sequence, charge state, and peptide ionmass-to-charge values are indicated. Acidic amino acids arehighlighted in red. (G) Relative intensities of LC-MS/MS traces of the heavy- and light-labeled correspondingunphosphorylated peptide, extracted to ±2 ppm. Peptide sequence, charge state, and peptide ion mass-to-charge values are indicated. Acidic amino acids are highlighted in red. Blue lines in (F) and (G) representthe samples incubated with buffer and gray lines represent the samples incubated with purified CK2.
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assay (fig. S8A). We confirmed the composition of the purified condensinI complex and PP6 holoenzymes byLC-MS/MS (table S5).We detected theendogenous inhibitors a4 and TIPRL (5, 65, 66) and the PPP6R and ANRsubunits with PP6c (fig. S8B). This is consistent with the observation thatfree catalytic PPP subunits are unstable and rapidly degraded in cells andrequire stabilization through binding to an endogenous inhibitor or regu-latory and scaffolding subunits (67).

In HeLa cells, condensin I subunit and PP6c protein abundance arecomparable (40). We incubated the purified condensin I complex with orwithout the purified PP6 holoenzyme at a 1:20 enzyme/substrate ratio(Fig. 5A). We used this high enzyme/substrate ratio because we expectedthat much of the purified PP6c was inactive due to binding to endogenousinhibitors (fig. S8B). After quenching the in vitro reactions, we resolvedthe proteins by SDS–polyacrylamide gel electrophoresis (SDS-PAGE),excised the bands corresponding to NCAP-G from the gel (Fig. 5B), anddigested the protein into peptides.After labeling the peptides using reductivedimethyl labeling chemistry, we mixed the labeled peptides and performedLC-MS/MS (Fig. 5A). We observed a strong decrease in NCAP-G phos-phorylation on Ser973/5 upon PP6c addition and an increase in the corre-sponding unphosphorylated peptide (Fig. 5, C and D, and table S6),whereas the doubly phosphorylated peptide was unchanged (table S6).Furthermore, another phosphorylation site on NCAP-G, Ser674, which alsocontains an acidophilic motif but was unchanged in PP6c-depleted cells(table S2), was also not dephosphorylated in vitro with purified PP6 (tableS6). Thus, we concluded that PP6 holoenzymes specifically dephosphorylatepSer973 or pSer975 on NCAP-G but only when singly phosphorylated in mi-totic HeLa cells and in vitro.


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The two candidate PP6 substrate siteson NCAP-G, Ser973 and Ser975, are sur-rounded by acidic amino acids (Fig. 5C),which is similar to the substrate motif pref-erence of CK2 (51). To establish that Ser973/5 is a CK2 phosphorylation site, we per-formed an in vitro kinase assay with CK2and purified condensin I subunits previ-ously dephosphorylated with nonspecificl-phosphatases, and identified phosphory-lated peptides by LC-MS/MS (Fig. 5Eand table S7). We observed an increase inSer973/5 phosphorylation and a decrease inthe corresponding unphosphorylated pep-tide in the samples exposed to CK2 (Fig.5, F and G). We did not observe the gener-ation of pSer973:975 in the CK2 reaction.Thus, these results indicated that CK2 islikely responsible for the single phospho-rylation of either Ser973 or Ser975 onNCAP-G and that PP6c dephosphorylatesthis site in mitotic cells.

Defects in chromosomecondensation and segregationupon PP6c depletionCells begin to condense their chromo-somes when they enter mitosis; this pro-cess continues throughout prometaphase.If mitotic progression is halted and cellsare arrested in prometaphase by spindlepoisons, condensin complex activity con-tinues in the arrested cells, which results

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in gradual hypercondensation of chromosomes (Fig. 6A). Disruption of thecondensin I complex by depletion of NCAP-D2 reduces this chromosomehypercondensation effect (70). To determinewhether depletion of NCAP-Ghad the same effect on chromosome condensation as depletion of NCAP-D2 and whether PP6c depletion mimics those effects, we depleted HeLacells of PP6c by infection with the viruses encoding PP6c-sh1 and PP6c-sh4,treated the cells with nocodazole (which arrests cells in prophase), performedchromosome spread assays, and analyzed the DNA for hypercondensation bymicroscopy (Fig. 6A). Indeed, we observed a similar decrease (~30%) in chro-mosome hypercondensation and a corresponding increase in normallycondensed chromosomes in chromosome spreads from cells depleted ofNCAP-G or PP6c compared to cells infected with the control virus (Fig. 6B).

Additionally, reduced condensin I function can result in chromosomesegregation errors, including lagging chromosomes and chromatinbridges (Fig. 6C) (71).We hypothesized that PP6-dependent dephospho-rylation of condensin I may prevent such chromosome segregation errorsin mitosis. We depleted HeLa cells of PP6c or NCAP-G and imaged an-aphase cells by fluorescencemicroscopy.Whereas NCAP-G knockdownresulted in a significant increase in both chromatin bridges and laggingchromosomes, PP6c depletion only produced a significant increase inchromatin bridges (Fig. 6D). Thus, as previously observed for NCAP-D2 (70, 71), NCAP-G is necessary for chromosome condensation uponprolonged prometaphase arrest as well as accurate chromosome segre-gation. Furthermore, depletion of PP6c results in similar defects in hy-percondensation, suggesting that the CK2-dependent phosphorylationsites on NCAP-G and potentially other members of the condensin Icomplex reduces the activity of the complex in mitotic PP6c-depleted cells.

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NCAP-G or PP6c depletion on chromosome hypercondensation. Immunofluorescence micrographs de-picting representative hypercondensed or normally condensed chromosomes in chromosome spreadsof HeLa cells. Scale bar, 10 mm. (B) Quantification of differences between normally condensed and hy-percondensed chromosomes upon NCAP-G depletion (left) and PP6c depletion (right). (C) Effect ofNCAP-G or PP6c depletion on the formation of lagging chromosomes and chromosome bridges. Immu-nofluorescence micrographs depicting representative images of chromosome segregation defects inanaphase in PP6c shRNA–infected HeLa cells. Scale bar, 5 mm. (D) Quantification of the number oflagging chromosomes and chromatin bridges in NCAP-G–depleted (left) or PP6c-depleted (right) HeLacells. Quantified data are shown as means with SD. **P < 0.005; *P < 0.05, paired Student’s t test (n = 3independent experiments).





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DISCUSSION

Here, we developed a baculovirus-mediated shRNA approach and com-bined it with quantitative phosphoproteomics to determine changes inphosphorylation and protein abundance upon depletion of PP6c. We chosea baculovirus-mediated gene silencing approach because recombinantbaculoviruses are safe, stable for long-term storage, amenable to ampli-fication, and can infect awide range of mammalian cells at high efficiency(34, 35). Because of these characteristics, baculoviruses are ideal for ef-ficient and reproducible gene depletion in large numbers necessary forlarge-scale phosphoproteomics experiments. We identified two shRNA se-quences that in combination efficiently reduced PP6c abundance andincreased PP6c substrate phosphorylation. However, as with other gene si-lencing approaches, potential off-target effects are a concern. Thus,weglob-ally compared protein abundance in mitotic HeLa cells upon infection witheach of these shRNAs individually.We found that the abundance of only 35proteins was statistically different between infections with the differentshRNAs, and the median fold change of this difference was only modest.Although statistically significant because of highly reproducible foldchanges in each triplicate analysis, we predicted that the minor absolute dif-ferences in the protein abundance of the 35 proteins were not likely biolog-ically important in the context of the large increases in phosphorylationoccupancy we observed in our combined hairpin shPP6 data set. Althoughwe cannot rule out that these hairpins may exhibit off-target effects on proteinsnot among the 6700 in this experiment, the lack of any compelling off-targetcandidates among this relatively deep proteomics data set is supportiveof their selectivity for PP6c.

Using this strategy, we identified 408 phosphopeptides on 272 proteinsthat increased and 298 phosphopeptides on 220 proteins that decreasedupon PP6c depletion in mitotically arrested HeLa cells. Characterizationof the motifs surrounding the phosphorylation sites that increased uponPP6c depletion identified an arginine-containing basophilic motif, whichis reminiscent of the substrate motif of the known PP6c substrate AURKA(21). Comparison with previously identified AURKA substrates (44) re-vealed that phosphorylation of only a subset of AURKA substrates in-creased upon PP6c depletion and that not all basophilic PP6c-dependentphosphorylation sites are increased because of higher AURKA activity.One explanation for this involves the relatively high phosphorylation siteoccupancy of mitotic phosphoproteins that may preclude some AURKAsubstrates from increasing in site occupancy any further upon PP6c deple-tion (40, 41). This may also reflect the complex structural and regulatoryrequirements for kinase activation that extend beyond activation T-loopphosphorylation status, in which AURKA may exhibit increased activitytoward select substrates. In addition, other phosphatases that directly tar-get AURKA substrates may exhibit differential activity on these substratesto tightly regulate their occupancy. Furthermore, although some Arg-X-phospho-Ser–containing phosphopeptides might be increased upon PP6cdepletion due to increasedAURKAactivity, it is possible that othersmightbe directly affected by PP6c depletion through opposition of AURKA orother basophilic kinases. Alternatively, the analysis of AURKA inhibitor–sensitive phosphorylation sitesmay have not identified changes in phospho-rylation site occupancy of some of these PP6c-specific phosphopeptidesbecause of experimental conditions, including short-term inhibition ofkinase activity (30 min) that might not be sufficient to allow for phospho-rylation site turnover by opposing protein phosphatases. Finally, there aremany other basophilic protein kinases that could play a role in regulatingthese sites, such as CHK2, calcium and calmodulin–dependent kinase II(CaMKII), protein kinase A (PKA), protein kinase B (PKB), and serum-and glucocorticoid-regulated kinase (SGK) (50). These differences in re-sponses of certain phosphorylation sites to selective kinase and opposing

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phosphatase inhibition further serve to highlight the difficultly in studyingthe posttranslational mechanisms that regulate systems as complex as celldivision.We only observed an enrichment for acidophilic sites in the por-tion of phosphorylation sites that were increased in abundance upon PP6cdepletion. This motif is similar to the substrate preference of CK2 (50–52),which has awide array of substrates and is an important regulator inmany sig-nalingpathways (72); however, its role inmitosis is just emerging (68,73–76).

Network analysis of proteins containing the regulated phosphoryl-ation sites revealed known and new biological processes and pathwaysthat are potentially directly or indirectly regulated by PP6. The identificationof PP6c-regulated phosphorylation sites on proteins linked to chromo-some condensation is intriguing. Protein phosphorylation is an importantregulatory mechanism in chromosome condensation, and the integrationof specific kinase activities in both interphase and mitosis is important forthe fidelity of chromosome condensation (69, 77–82). Furthermore, the roleof protein phosphatases in chromosome condensation and decondensa-tion is just emerging (83–85). We identified several phosphorylationsites on members of the condensin I complex that increased upon PP6c de-pletion. Condensin I is a pentameric protein complex, containing NCAP-G,NCAP-H, NCAP-D2, SMC2, and SMC4, that associates with chromo-somes in prometaphase after nuclear envelope breakdown (82). CondensinI is required for longitudinal chromosome compaction in prolonged pro-metaphase, complete dissociation of cohesin from chromosome arms,and normal timing of mitotic progression (70). Furthermore, condensin Iis important for mechanical rigidity and assembly of mitotic chromosomesand faithful chromosome segregation. Depletion of condensin I subunitsleads to defects in anaphase, including the formation of chromatin bridgesand lagging chromosomes (71). In vitro, condensin I introduces positivesupercoils into closed circular DNA in an adenosine triphosphate (ATP)–dependent manner (86). CK2 phosphorylates and thereby negatively regu-lates condensin I function in interphase by decreasing the supercoilingactivity of condensin I (81); this is thought to prevent premature or inap-propriate chromosome condensation outside of mitosis. As cells enter mito-sis, condensin I is phosphorylated by Cdk1, which targets condensin I tochromosomes and partially activates it (81). To achieve full condensin I ac-tivation as cells proceed through mitosis, the CK2-phosphorylated sites oncondensin I must be dephosphorylated relative to their status in interphase(81). This reduced phosphorylation could either result from a change inCK2 activity or substrate accessibility, or from increased activity of a proteinphosphatase, or both. On the basis of the CK2 consensus motif, previous re-search suggested that CK2 phosphorylates Thr951 on SMC4, Ser1371 on

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anism of condensin I regulation adapted from Takemoto et al. (81). In G1/S,condensin I is phosphorylated by CK2, maintaining condensin I in an in-active state. In G2, Cdk1 phosphorylates condensin I, which partially acti-vates the DNA supercoiling activity of the complex. As cells enter mitosisand duringmitosis, CK2-dependent phosphorylation sites become dephos-phorylated, maximally activating condensin I. On the basis of our results,PP6c contributes to this process by dephosphorylating CK2 sites onNCAP-G.





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NCAP-D2, Ser975 on NCAP-G, and Ser570 on NCAP-H (81). However, thiswas only confirmed experimentally for Ser570 on NCAP-H (81). Of the fourpotential CK2 phosphorylation sites in proteins in the condensin I complex[Thr951 on SMC4, Ser1371 on NCAP-D2, Ser975 on NCAP-G, and Ser570 onNCAP-H (81)],weonlydetectedpeptidescoveringSer973/5onNCAP-G; insilicosequence analysis revealed thatmany of these potential CK2 phosphorylationsites are in highly acidic, lysine- and arginine-free regions of protein sequencethat are not accessible by trypsin digestion. We confirmed that NCAP-GSer973/5 is a direct substrate of PP6c by in vitro phosphatase assays, and datafrom invitro kinase assays showed that PP6c opposes CK2 phosphorylationon the NCAP-G, indicating that PP6c could contribute to condensin I acti-vation through dephosphorylation of CK2 phosphorylation sites (Fig. 7).

Inhibition of condensin I function by subunit depletion results inchromosome condensation defects in cells experiencing prolonged mitoticarrest and chromosome segregation defects in cells in anaphase (70, 71).We found that depletion of PP6c resulted in chromosome condensation andsegregation defects similar to those observed with depletion of NCAP-G.Given that we were unable to profile other known CK2-dependent sites oncondensin complex subunits in our large-scale PP6c screen, possibly be-cause of their inaccessibility by trypsin-mediated proteolysis, we recognizethat not all of the inhibitory function of condensin I is likely to be regulatedthrough dynamic phosphorylation of Ser973/5 on NCAP-G. However, weestablished that CK2 can mediate NCAP-G phosphorylation at Ser973/5

and that PP6 dephosphorylates Ser973/5, thereby opposing this activity ofCK2. In vitro, CK2 did not generate pSer973:975, suggesting that a differentprotein kinase may mediate the second phosphorylation event or thatpriming phosphorylation events (87) that were likely removed by previousl-phosphatase treatment are required forCK2processivity at this locus. PP6neither in cells nor in vitro dephosphorylated this locus when both siteswere phosphorylated, suggesting that the second phosphorylation sitemay function to inhibit PP6 activity here. Clearly, there are additional layersof complexity between PP6, CK2, and condensin I function that remain tobe explored.

Broadly speaking, the interaction and counteraction between proteinkinases and phosphatases are essential for cellular function and organismalsurvival; these delicate balances are often deregulated in human cancer. Ascells progress through mitosis, they undergo marked changes in cellularstructure and organization to successfully divide. These changes are regu-lated and controlled by dynamic protein phosphorylation to ensure faithfulchromosome segregation and distribution of cellular content (1, 2). Al-though the importance of protein phosphatases is well recognized, theidentification of candidate protein phosphatase substrates is hindered bythe lack of small molecule inhibitors that specifically target the catalyticsubunit of a single PPP family member. Thus, whereas the approach thatwe present cannot distinguish direct substrates of the phosphatase fromindirect effects of phosphatase depletion, it provides valuable insights intophosphatase biology and pathways and provides another strategy for re-vealing previously unknown protein kinase—phosphatase interactions inthe regulation of phosphorylation site occupancy of common substrates.

MATERIALS AND METHODS

CellsHeLa cellswere grown as adherent cultures inDulbecco’smodified Eagle’smedium (Cellgro Mediatech Inc.) with 10% heat-inactivated fetal bo-vine serum (FBS) (HyClone) and penicillin-streptomycin (100 U/ml and100 mg/ml, respectively; Cellgro Mediatech Inc.) at 37°C in a humidifiedincubator with 5% CO2. Sf9 cells were grown in Grace’s supplemented in-sect cellmedium (LifeTechnologies)with 10%heat-inactivatedFBS (HyClone),

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gentamicin (10 mg/ml; Sigma), and amphotericin B (0.25 mg/ml; Sigma).Sf9 cells were maintained at 27°C in a nonhumidified incubator.

Recombinant baculovirus-based shRNA generationTo generate recombinant baculovirus expressing shRNAs, the polyhedrinpromoter was deleted from pFastBac1 (Life Technologies). A fragmentof the pLL3.7 lentivirus vector (88) containing the U6 promoter, shRNA,CMV promoter, and EGFP complementary DNA was subcloned intopFastBac1 to generate pFastBacMam-shRNA-GFP. shRNA sequences tar-getingPP6cwere chosenwith the siDESIGNCenter (Dharmacon) and fromZeng et al. (21): PP6c-1 (GTTTGGAGACCTTCACTT), PP6c-2 (CGCTA-GACCTGGACAAGT), PP6c-3 (GTAAATACAAGAGAACCA), PP6c-4(CTAAATGGCCTGATCGTA), and PP6c-5 (GTAGACAGAGGTTACTAT).Baculoviruses of PP6c-1 and PP6c-4 were efficient in reducing PP6c ex-pression. Specificity for PP6c was confirmed using BLAST search againstthe human genome. shRNA sequenceswere cloned into pLL3.7, subclonedinto the modified pFastBac1, and confirmed by DNA sequencing.Sequence-confirmed vectors were transformed in DH10Bac (Life Technol-ogies) to generate bacmids. Recombinant baculoviruses were generatedaccording to themanufacturer’s instructions. Viruseswere further amplifiedin Sf9 insect cells.

shRNA testingHeLa cells were infected with P0 baculovirus. Infection efficiency wasdetermined by monitoring GFP expression 24 hours after infection. Deple-tion of PP6cwasmonitored at 24, 48, and 72 hours after infection byWesternblotting with a PP6c-specific antibody (Abcam). For Western blot analysis,cellswere collected,washedwith phosphate-buffered saline (PBS) (pH7.4),and lysed in lysis buffer [50 mM tris-HCl (pH 8.1), 150 mM NaCl, 15%glycerol, 1% SDS, phosphatase inhibitors, protease inhibitors, 1 mMdithiothreitol (DTT)].

TMT quantitative proteomicsHeLa cells treated with control, shPP6c-1, or shPP6c-4 baculovirus weresynchronized and arrested in mitosis in biological triplicate.Mitotic cellswere collected by shake-off, washed twice with PBS, pelleted, and lysedin 9 M urea, 50 mM tris (pH 8.2), 100 mM NaCl buffer in the presenceof protease inhibitors by sonication. A 50-ml aliquot of the lysate was re-moved for quantification by bicinchoninic acid assay (BCA) (Pierce); theremainder of the lysate was reduced with 5 mM DTT at 50°C for 25 min,followed by alkylation with 13 mM iodoacetamide at room temperature for1 hour. The lysates were then diluted fivefold with 50 mM tris (pH 8.2) and75mMNaCl and digested with trypsin (1:100w/w) for 16 hours at 37°C.The digests were desalted using C18 solid-phase extraction cartridges(Grace-Vydac); an aliquot of each of the desalted eluates correspondingto 50 mg of peptide digest was dried by vacuum centrifugation in sepa-rate tubes.

The nine samples were prepared for TMT-based quantitative proteo-mics as one sixplex (biological replicates 1 and 2) and one triplex(biological replicate 3). Previously dried, individual TMT reagent ali-quots corresponding to 133 mg of reagent were resuspended in 40 mlof 150mMHepes (pH 8.5)/20% acetonitrile (ACN), followed by transferof the resuspended reagent aliquots to tubes containing 50 mg of peptidedigest and vortexing to mix reagent and peptides. After 1 hour at roomtemperature, each digest was quenched with 5 ml of 500 mM ammoniumbicarbonate solution for 10 min, mixed into the two sets of multiplexes,diluted threefold with 0.1% trifluoroacetic acid (TFA) in water, and de-salted using C18 solid-phase extraction cartridges as above. The desaltedmultiplexes were dried by vacuum centrifugation, resuspended in 110 mlof 3% ACN/0.1% TFA in water, and separated on a pentafluorophenyl






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(PFP) analytical column [XSelect HSS PFP XP column, Waters; 100 A,2.5 mm inner diameter (ID), 150 mm in length] into 48 fractions by gra-dient elution [buffer A (3% ACN, 0.1% TFA); buffer B (95% ACN, 0.1%TFA)] with 5% B, 0 to 1 min; 5 to 11% B, 1 to 2 min; 11 to 47% B, 2 to60min; 47 to100%B,60 to61min; 100%B,61 to69min; 100 to 5%B, 69 to70 min; 5% B, 70 to 110 min, all at a flow rate of 0.15 ml/min. The 48fractionswere reduced to 24 bymixing fraction 1with fraction 13, fraction2 with fraction 14, etc., and dried by vacuum centrifugation. Basic pHreverse-phase system is as described below. The equivalent of 1.5 mg of pep-tide from each fraction was analyzed using an Easy LC-1000 (Proxeon) andOrbitrap Fusion (89) (Thermo Fisher Scientific) LC-MS/MS platform acrossa 2-hour gradient from9%ACN/0.1% formic acid to 37%ACN/0.1% formicacid. The Orbitrap Fusion was operated in data-dependent, SPS-MS3 quan-tificationmode (38, 90) wherein anOrbitrapMS1 scanwas taken [scan range,350 to 1500mass-to-charge ratio (m/z); resolution (R), 120K; automatic gaincontrol (AGC) target, 2.5 × 105; max ion injection time, 100 ms], followedby ion trap MS2 scans on the most abundant precursors for 4 s [max speedmode; quadrupole isolation, 0.6m/z; AGC target, 4 × 103; scan rate, rapid;max ion injection time, 60 ms; minimum MS1 scan signal, 5 × 105 nor-malized units; charge states 2, 3, and 4 included; collision-induced disso-ciation (CID) energy, 33%] and Orbitrap MS3 scans for quantification [R,15K; AGC target, 2 × 104; max ion injection time, 125 ms; higher-energycollisional dissociation (HCD) energy, 48%; scan range, 120 to 140 m/z;synchronous precursors selected, 10). The resulting data fileswere searchedusingCometwith a staticmass of 229.162932 daltons on peptideN-terminiand lysines and 57.02146 daltons on cysteines and a variable mass of15.99491 daltons onmethionines against the target-decoy version of the hu-man proteome FASTA (UniProt) and filtered to a ~1% FDR at the peptidelevel. TMTreporter ionswere filtered to require aminimum signal of 3000U,and at least two unique peptide sequences were required to contain quan-tification results per protein. These requirements resulted in 6700 proteinquantifications across the two multiplexes (1.2% protein FDR by target-decoy. To assess differences in protein abundance between the two shRNAs,we required protein quantifications in both hairpins to be statistically signif-icant (P < 0.05, two-tailed Student’s t test), and assessed differences be-tween the two hairpins from those protein abundances (P < 0.05, two-tailedStudent’s t test).

Proteome-wide analysis of PP6c depletionHeLa cells were seeded at 15% confluency and infected with PP6c-sh1–and PPc-sh4–expressing virus or control shRNA–expressing virus thenext day. Twenty-four hours after infection, HeLa cells were synchro-nized at the G1/S transition by the addition of thymidine (final concentra-tion, 2 mM; Sigma) for 16 hours followed by a washout for 3 hours andaddition of Taxol (100 nM, Sigma) for 16 hours. For thymidine washout,cells werewashed three timeswith 15ml of PBS (CellgroMediatech Inc.).Mitotic HeLa cells were collected bymitotic shake-off, washed with PBS,snap-frozen in liquid nitrogen, and stored at −80°C. Efficiency of PP6c de-pletion and increased phosphorylation of Thr288 ofAURKAwere determinedby Western blotting with specific antibodies against PP6c (Abcam) andAURKAThr288 (Cell SignalingTechnologies).Western blot intensitieswerequantified using ImageJ (91). HeLa cell pellets were thawed on ice, lysed inice-cold lysis buffer [8 M urea, 25 mM tris-HCl (pH 8.6), 150 mM NaCl,phosphatase inhibitors (2.5 mM b-glycerophosphate, 1 mM sodium fluo-ride, 1 mM sodium orthovanadate, 1 mM sodiummolybdate), and proteaseinhibitors (1 mini-Complete EDTA-free tablet per 10 ml of lysis buffer;Roche Life Sciences)], and sonicated three times for 15 s each with inter-mittent cooling on ice. Lysates were centrifuged at 15,000g for 30 min at4°C. Supernatants were transferred to a new tube, and the protein concen-tration was determined using a BCA assay (Pierce/Thermo Fisher Scien-

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tific). For reduction, DTTwas added to the lysates to a final concentrationof 5mMand incubated for 30min at 55°C. Afterward, lysateswere cooledto room temperate and alkylated with 15mM iodoacetamide at room tem-perature for 45 min. The alkylation was then quenched by the addition ofan additional 5mMDTT.After sixfold dilutionwith 25mM tris-HCl (pH8),the samples were digested overnight at 37°C with 2.5% (w/w) trypsin. Thenext day, the digest was stopped by the addition of 0.25% TFA (final, v/v)and centrifuged at 3500g for 30 min at room temperature to pellet precipi-tated lipids, and peptides were desalted on a 500-mg (sorbent weight) solid-phase extractionC18 cartridge (GraceDavison).Aliquots of peptide samples(250 mg each) were taken for protein abundance analysis. Peptides werelyophilized and stored at −80°C until further use.

Phosphopeptide enrichmentPhosphopeptide purification was performed as previously described (92).Briefly, peptideswere resuspended in 2M lactic acid in 50%ACN(“bindingsolution”). Titanium dioxide microspheres were added and vortexed byaffixing to the top of a vortexmixer on the highest speed setting at room tem-perature for 1 hour. Afterward, microsphereswerewashed twicewith bindingsolution and three times with 50% ACN/0.1% TFA. Peptides were elutedtwice with 50 mMKH2PO4 (adjusted to pH 10 with ammonium hydroxide).Peptide elutions were combined, quenched with 50% ACN/5% formic acid,dried, and desalted on a mHLB OASIS C18 desalting plate (Waters).

Dimethyl labelingDimethyl labeling was performed essentially as described (93). Peptidesand phosphopeptides were resuspended in 100 mM triethyl ammoniumbicarbonate (TEAB) by vortexing. For “light” labeling, formaldehyde(final concentration of 0.25%) and cyanoborohydride (final concentra-tion of 25 mM) were added to the samples followed by vortexing. For“heavy” labeling, formaldehyde–13C d2 (final concentration of 0.25%)and cyanoborodeuteride (final concentration of 25 mM) were added tothe sample followed by vortexing. Samples were incubated at room tem-perature for 1 hour. Afterward, reactions were quenched by the additionof NH3 solution (final concentration of 0.2%), followed by vortexingand incubation for 5 min at room temperature. Reactions were furtherquenched by the addition of formic acid (final concentration of 0.5%),followed by vortexing and incubation for 5 min at room temperature.Heavy and light samples were mixed, acidified with TFA (final concen-tration of 0.1%), and desalted.

SCX chromatographyPhosphopeptides were resuspended in SCX buffer A [7 mM KH2PO4

(pH 2.65)/30% ACN] and separated per injection on an SCX column(Phenomenex Luna SCX; 2.1-mm ID × 200-mm length) as previouslydescribed (92) and under the following conditions: using a gradient of 0 to10% SCX buffer B [350 mM KCl/7 mM KH2PO4 (pH 2.65)/30% ACN]for 10 min, 10 to 17% SCX buffer B for 17 min, 17 to 32% SCX buffer Bfor 13min, 32 to 60%SCX buffer B for 10min, 60 to 100%SCX buffer Bfor 2 min, holding at 100% SCX buffer B for 5 min, from 100 to 0% SCXbuffer B for 2 min, and equilibration at 0% SCX buffer B for 65min, all ata flow rate of 0.2 ml/min, after a full blank injection of the same programwas run to equilibrate the column. Sixteen fractions were collected, dried,and desalted using a mHLB OASIS C18 96-well desalting plate and mani-fold (Waters).

Basic pH reversed-phase high-performanceliquid chromatographyPeptides were resuspended in buffer A [5% ACN, 10 mM ammoniumbicarbonate (pH 8.0)] and separated per injection on an Agilent 300 Extend

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C18 column (5-mm particles, 4.6-mm ID, and 200-mm length) using a45-min linear gradient from 8 to 35%ACN (for dimethyl-labeled peptides)and from11 to 39%ACN (for TMT-labeled peptides) in 10mMammoniumbicarbonate (pH 8) on an Agilent 1100 liquid chromatography system.Peptides were separated into 96 fractions. The 96 fractionswere combinedinto 12 samples (for dimethyl-labeled peptides) and 24 samples (for TMT-labeled peptides), dried, and desalted using a mHLB OASIS C18 96-welldesalting plate and manifold (Waters).

LC-MS/MS AnalysisLC-MS/MS analysis for peptides and phosphopeptides was performed ona Fusion Orbitrap Tribrid mass spectrometer (Thermo Fisher Scientific)equipped with an Easy-nLC 1000 (Thermo Fisher Scientific) and nano-spray source (Thermo Fisher Scientific). Phosphopeptides were redissolvedin 5% ACN/1% formic acid and loaded onto a trap column at 2500 nl/min[1.5-cm length, 100-mmID;ReproSil,C18AQ5-mm200-Åpore (Dr.Maisch)]vented to waste through a micro-tee and eluted across a fritless analyticalresolving column (35-cm length, 100-mm ID; ReproSil, C18 AQ 3-mm200-Å pore) pulled in-house (Sutter P-2000, Sutter Instruments) with a60-min gradient of 5 to 30% LC-MS buffer B (LC-MS buffer A: 0.0625%formic acid, 3% ACN; LC-MS buffer B: (0.0625% formic acid, 95% ACN).TheOrbitrapFusionwas set to performanOrbitrapMS1 scan (R, 120K;AGCtarget, 2.5 × 105) from350 to 1500Thomson, followed by HCDMS2 spec-tra on the most abundant precursor ions detected by Orbitrap scanning(R, 15K; AGC target, 40,000; max ion time, 50 ms) for 2.5 s before re-peating the cycle. Precursor ions were isolated for HCD with a quadru-pole isolation width of 0.6 Thomson and HCD fragmentation at 30%collision energy. Charge state 2, 3, and 4 ions were selected for MS2.

Peptide spectral matching and bioinformaticsRaw datawere searched using SEQUEST (94, 95) (Thermo Fisher Scien-tific) against a target-decoy (reversed) (96) version of the human proteomesequence database (UniProt; downloaded February 2013) with a precursormass tolerance of ±1 dalton and requiring fully tryptic peptides with up tothree miscleavages. Carbamidomethylcysteine, dimethylation at peptideN-termini, and lysines were enabled as fixedmodifications. Oxidized me-thionine; phosphorylated serine, threonine, and tyrosine; and isotopicallyheavy label (+8.04437) at peptide N-termini and lysines were enabled asvariable modifications. The resulting peptide spectral matches were filteredto <1%FDR for peptides and 2%FDR for phosphopeptides, on the basis ofreverse-hit counting [mass measurement accuracy cutoffs within ±2.5 ppm,a d-XCorr (dCn) of greater than 0.08, and appropriate XCorr values). Prob-ability of phosphorylation site localization was determined by phosphoRS(97). Peptide ratio quantification was performed using MassChroQ (98).Log2 H/L ratios were median-adjusted for mixing errors. Phosphopeptidefold changes were adjusted for changes in protein abundance on a per-sample basis. Significance of log2 protein-corrected phosphopeptide foldchange was determined by two-tailed Student’s t test assuming unequalvariance. For further analysis, we required an at least twofold increase atP < 0.1. Peptide motif analysis was performed on phosphopeptides in-creased by twofold or more and a P value of <0.1 based on (42). Protein-protein interactions of proteins belonging to phosphopeptideswith significantincrease in phosphorylation occupancy were determined using the STRINGdatabase and analyzed in Cytoscape (54, 55). Edges present protein-proteininteractions based on the STRING database. Densely connected clusterswith the networks were identified using ClusterONE (99) in Cytoscape(minimum size, 4; node penalty, 2.4; overlap threshold, 0.5). GO annotationswere performed inCytoscape usingBiNGO to test for ontology enrichment.To determine significance of enrichment of terms, a Bonferroni-correctedPvalue cutoff of 0.05 was used.

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PP6 in vitro phosphatase assay293T cells were transfected with p3×Flag-CMV10-PP6c or p3×Flag-CMV10-NCAP-H, and stable cells lines were selected using G418. PP6choloenzymes and condensin I complex were purified separately using anti–FlagM2 affinity gel (Sigma) (15 ml of resin for each 15-cm tissue culture dishlysed) and eluted with 3×Flag peptide (final concentration, 150 ng/ml). Fordephosphorylation reactions, condensin I was incubated in the presence orabsence of PP6c holoenzymes in phosphatase buffer [50 mM Hepes (pH 7.5),10mMNaCl, 2mMDTT, 1mMMnCl2, 0.01%Brij 35] and incubated for2 hours or overnight at room temperature. We found that NCAP-G was ef-ficiently dephosphorylated after 2 hours, andwe did not observe any furtherdephosphorylation of other phosphorylation sites on NCAP-G in theovernight reactions. Reactions were quenched by the addition of SDS-PAGEsample buffer, reduced, and alkylated (as described above), and then separatedby SDS-PAGE gel electrophoresis. To better access the region of NCAP-Gcorresponding to Ser973 and Ser975, bands corresponding to NCAP-G wereexcised and digestedwith proteinaseK in 50mMTEAB for 1 hour at 37°C.Peptides were extracted using 5% formic acid/50% ACN and dried. Pep-tides were labeled heavy and light by reductive dimethylation (as describedabove), mixed, and desalted. Peptides were analyzed on a Q-Exactive Plusmass spectrometer (Thermo Fisher Scientific) or Orbitrap Fusion massspectrometer equipped with an Easy-nLC 1000 (Thermo Fisher Scientific).Raw data were searched using COMET in high-resolution mode (100)against a NCAP-G sequence database with a precursor mass tolerance of±20 ppm, no enzyme specificity, and carbamidomethylcysteine, and di-methylation at peptide N-termini, and lysines enabled as fixed modif-ications. Oxidized methionine; phosphorylated serine, threonine, andtyrosine; isotopically heavy label (+8.04437) at peptide N-termini andlysines were searched as variable modifications. Probability of phospho-rylation site localization was determined by phosphoRS (97). Quantifi-cation of LC-MS/MS spectra was performed using MassChroQ (98).Reproducibility of proteinase K digest is depicted in fig. S9, and resultsare shown in table S8.

CK2 in vitro kinase assayCondensin I was purified from 293T cells expressing p3×Flag-CMV10-NCAP-H as described above and dephosphorylated with 200 U of l-phosphatase (New England Biolabs). l-Phosphatase was inhibited by theaddition of 5 mM sodium vanadate, and condensin I was incubated with125 U of CK2 (New England Biolabs) in protein kinase buffer (New EnglandBiolabs) supplemented with 200 mM ATP (Sigma) at 30°C for 1 hour.Reactions were quenched by the addition of SDS-PAGE sample buffer,reduced, and alkylated (as described above), and then separated bySDS-PAGE gel electrophoresis. Bands corresponding to NCAP-G wereexcised and analyzed by proteinase K digest and reductive dimethyllabeling as described above, as well as by LC-MS/MS.

Analysis of mitotic defectsHeLa cells were seeded on glass coverslips and infected with virusesexpressing PP6c-sh1 and PP6c-sh4 or viruses expressing control shRNAfor 48 hours. Cells were fixed in 3.7% formaldehyde for 7 min at roomtemperature, permeabilized with PBS containing 0.1% Triton X-100(PBST), and blocked with 3% bovine serum albumin (BSA) in PBSTfor 30 min at room temperature. Afterward, cells were incubated withprimary antibodies against CRESTautoimmune serum (ImmunoVision)and a-tubulin (Sigma) diluted in 3%BSA in PBST for 30min at room tem-perature. Cells were washed with PBST and incubated with anti-humanAlexa 568 and anti-mouse Alexa 488 (Molecular Probes) secondary anti-bodies diluted in 3%BSA inPBST for 30min at room temperature.DNAwaslabeledwithNucBlue FixedCell ReadyProbes reagent (LifeTechnologies).




Images were collected on an Axioplan2 Zeiss fluorescence microscope.Experiments were done in biological triplicates. At least 100 HeLa cells inanaphase were counted per condition per experiment.

Chromosome spreadsHeLa cells were infected with PP6c or control shRNA for 48 hours andtreated for 4 hours with nocodazole (100 ng/ml). Cells were collected bymitotic shake-off and pelleted. Cells were resuspended gently in 75 mMKCl, incubated at room temperature for 5 min and at 4°C for 1 min, andpelleted by centrifugation. Cells were fixed with methanol/acetic acid (3:1)and pelleted. Cells were washed twice with methanol/acetic acid (3:1), re-suspended, and dropped onto glass slides. Slides were allowed to dry, andDNAwas stained with NucBlue Fixed Cell ReadyProbes reagent (Life Tech-nologies) diluted in PBS for 30min and sealedwith coverslips using ProLongGold (Life Technologies). Experimentswere done in biological triplicates. Atleast 100 chromosome spreads were counted per condition per experiment.

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SUPPLEMENTARY MATERIALSwww.sciencesignaling.org/cgi/content/full/8/398/rs12/DC1Fig. S1. Characterization of PP6c shRNAs.Fig. S2. Quantitative comparison of protein abundance changes upon PP6c depletion withindividual PP6c shRNAs.Fig. S3. Scheme of cell cycle synchronization.Fig. S4. Characterization of PP6c-regulated phosphopeptides.Fig. S5. LC-MS/MS trace and MS/MS spectra of known PP6c substrate DNA-PK pSer3205.Fig. S6. Interaction network of proteins with increased and decreased phosphorylationupon PP6c depletion.Fig. S7. LC-MS/MS trace and MS/MS spectra of NCAP-G pSer973/5.Fig. S8. Characterization of PP6c and NCAP-G purifications.Fig. S9. NCAP-G analysis.Table S1. Table containing TMT protein quantification results.Table S2. Table containing significantly increased phosphopeptides due to phosphorylation orprotein abundance, significantly decreased phosphopeptides due to phosphorylation or pro-tein abundance, and all phosphopeptide and protein data.Table S3. Table containing quantification results of AURKA substrates.Table S4. Table containing quantification results of RXS motif–containing phosphopeptidesthat increase in phosphorylation occupancy upon PP6c depletion.Table S5. Table containing proteins identified in 3×Flag-NCAP-H and 3×Flag-PP6c purification.Table S6. Table containing quantification results of the PP6c in vitro phosphatase assaysof NCAP-G.Table S7. Table containing quantification results of the CK2 in vitro kinase assay ofNCAP-G.Table S8. Table containing quantification results of proteinase K digest of NCAP-G.

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Acknowledgments: We thankmembers of the Kettenbach laboratory for helpful discussion.We would like to thank J. Gui for guidance with the statistical analyses performed, andL. Kabeche for help with setting up chromosome spreads. Funding: Funded by theAmerican Cancer Society Research Grant IRG-82-003-30 (A.N.K.). The Orbitrap FusionTribrid mass spectrometer was acquired with support from NIH (S10-OD016212). Authorcontributions:S.F.R. performed the PP6c shRNA phosphoproteomics and proteomics ex-periment and in vitro phosphatase assay and characterized the effects of PP6c and NCAP-Gdepletion on chromosome segregation and condensation. K.A.S. generated and character-ized the PP6c shRNA constructs. M.E.A. managed data files and the data pipeline for allproteomics experiments and generated software for iBAQ quantification and motif analy-sis. A.N.K. designed the study and performed the TMT protein quantification experiment.S.F.R. and A.N.K. analyzed the data and generated figures. A.N.K. wrote the manuscript.Competing interests: The authors declare that they have no competing interests. Dataand materials availability: The mass spectrometry proteomics data have been depositedto the ProteomeXchange Consortium (101) through the PRIDE (PRoteomics IDEntifications)partner repository. PX accession no. PXD002609.

Submitted 8 April 2015Accepted 24 September 2015Final Publication 13 October 201510.1126/scisignal.aab3138Citation: S. F. Rusin, K. A. Schlosser, M. E. Adamo, A. N. Kettenbach, Quantitativephosphoproteomics reveals new roles for the protein phosphatase PP6 in mitotic cells.Sci. Signal. 8, rs12 (2015).

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cellsQuantitative phosphoproteomics reveals new roles for the protein phosphatase PP6 in mitotic

Scott F. Rusin, Kate A. Schlosser, Mark E. Adamo and Arminja N. Kettenbach

DOI: 10.1126/scisignal.aab3138 (398), rs12.8Sci. Signal.

regulated by PP6 in mitotic cells.phosphorylation event in this critical complex and provided many other potential direct substrates of PP6 and pathwaysphosphorylated by casein kinase 2 and dephosphorylated by PP6. Thus, this study identified a mitotically regulated Biochemical analysis showed that a subunit of the complex necessary for chromosome condensation wasin phosphorylation identified roles for PP6 in the regulation of RNA splicing, rRNA processing, and translation. important for chromosome segregation and spindle formation, network analysis of the proteins that exhibited differencesmay oppose. In addition to the known role of PP6 in reducing the activity of the Aurora kinase A, a kinase that is Motif analysis of the sequences of the sites that changed in the PP6c-depleted cells suggested kinases that PP6 activitydepleting HeLa cells of the catalytic subunit of PP6 on the phosphoproteome when the cells were arrested in mitosis.

. analyzed the effect ofet alTo study the function of the serine-threonine protein phosphatase PP6, Rusin Exploring mitotic phosphatase function

ARTICLE TOOLS http://stke.sciencemag.org/content/8/398/rs12

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Quantitative phosphoproteomics reveals new roles for the ... · PP6-regulatedphosphorylationevents.Furthermore,wecharacterizedarole for PP6 in chromosome condensation byopposing casein