Phosphoproteomic Analysis of Aurora Kinase Inhibition in Monopolar

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Letter

Phosphoproteomic analysis of Aurora kinase inhibition in Monopolar Cytokinesis Ayse Nur Polat, Ozge Karayel, Sven H. Giese, Busra Harmanda, Erdem Sanal, Chi-Kuo Hu, Bernhard Y. Renard, and Nurhan Özlü J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.5b00645 • Publication Date (Web): 13 Aug 2015 Downloaded from http://pubs.acs.org on August 19, 2015

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ABSTRACT FIGURE

Cells grown in Lys0, Arg0 Cells grown in Lys8, Arg10

Purvalanol A

Purvalanol A and VX680 or AZD1152

C-phase + VX680 or AZD1152

C-phase Mock Lyse cells mixed 1:1

Tryptic digest

TiO2 phosphopeptide enrichment

LC-MS/MS

AURORA MOTIF

DNA/Tubulin/Vimentin

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Journal of Proteome Research

Control

+ AZD1152

ACS Paragon Plus Environment

+ VX680


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Phosphoproteomic

analysis

of

Aurora

kinase

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inhibition

in

Monopolar Cytokinesis

Ayse Nur Polat1, Özge Karayel1, Sven H. Giese2, 4, Büşra Harmanda1, Erdem Sanal1, Chi-Kuo Hu3, Bernhard Y. Renard2, Nurhan Özlü*1

1

Department of Molecular Biology and Genetics, Koç University, Istanbul, Turkey

2

Research Group Bioinformatics (NG 4), Robert Koch-Institute, Berlin, Germany.

3

Department of Genetics, Stanford University, School of Medicine, CA.

4

Department of Bioanalytics, Institute of Biotechnology, Technische Universität Berlin,

Berlin Germany *To whom correspondence should be addressed: Nurhan Özlü, PhD Department of Molecular Biology and Genetics Koç University Istanbul, Turkey email: [email protected] P: +90 212 338 1571 F: +90 212 338 1559

Running Title: Phosphoproteomic analysis of Aurora kinase inhibition


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Summary/Abstract

Cytokinesis is the last step of the cell cycle which requires coordinated activities of the microtubule cytoskeleton, actin cytoskeleton and membrane compartments. Aurora B kinase is one of the master regulatory kinases that orchestrate multiple events during cytokinesis. To reveal targets of the Aurora B kinase, we combined quantitative mass spectrometry with chemical genetics. Using the quantitative proteomic approach, SILAC (Stable Isotope Labeling with Amino acids in Cell culture), we analyzed the phosphoproteome of monopolar cytokinesis upon VX680 or AZD1152 mediated Aurora kinase inhibition. In total our analysis quantified over twenty thousand phosphopeptides in response to the Aurora-B kinase inhibition; 246 unique phosphopeptides were significantly down-regulated and 74 were up-regulated. Our data provide a broad analysis of downstream effectors of Aurora kinase and offer insights into how Aurora kinase regulates cytokinesis.

keywords: SILAC, Aurora kinase, VX680, AZD1152, mass spectrometry, phosphoproteome, cytokinesis



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INTRODUCTION:

Cytokinesis is the last step of the cell cycle in which a cell is divided into two after completing chromosome segregation. It requires coordinated activities of the microtubule cytoskeleton, actin cytoskeleton and membrane compartments 1. It is one of the shortest phases of the cell cycle displaying dramatic changes in cellular morphology and biochemistry. Cytokinesis events take place only for a short time window called C-phase, which is triggered by the exit of mitosis 2. During the Cphase, the actomyosin contractile ring assembles at the cortex midway between segregating chromosomes. The contraction of the actomyosin ring creates a cleavage furrow which forms a membrane barrier between the separating daughter cells 3.

Global approaches to the study of cytokinesis including traditional genetics and RNAi screens have been successful in providing a “parts list” of several proteins involved in cytokinesis 4. However, it has been more challenging to study the biochemistry of cytokinesis. One technical challenge is accessing an appropriate cell cycle stage due to the lack of a robust synchronization method for the C-phase. Arresting cells with an anti-microtubule drug followed by washout only gives partial C-phase cells mixed with mitotic cells, which makes it difficult to separate M- and C-phase. In a recent pharmaceutical method, instead of releasing into cytokinesis, cells were arrested in monopolar mitosis using kinesin-5 inhibitor S-trityl-L-cysteine followed by induction of C-phase using a CDK inhibitor purvalanol-A. Cytological characterizations of monopolar C-phase revealed new insights into midzone microtubule organization and midbody assembly during cytokinesis 5. In our previous study, we employed monopolar C-phase to provide the first broad overview of C-phase biochemistry 6. By


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combining SILAC and phosphopeptides enrichment methods, we quantitatively compared changes in the M-phase to C-phase transition 6. Our analysis revealed a major reorganization of microtubules during this transition; several mitotic spindle proteins lose their affinity to microtubules and midzone and +tip proteins gain affinity. Protein phosphorylation analysis in total cell lysate and in microtubule binding proteins revealed that there are more phosphorylation events in M-phase than C-phase. In the M-phase, the dominant motif was, as expected, the known Cdk1 consensus motif. In the C-phase, a motif that is similar to CDK1 still dominated but Aurora-B kinase was the second notable consensus sequence listed. Using a small molecule kinase inhibitor of Aurora kinase, VX680 7, this study further showed that Aurora kinases perturbed C-phase microtubule binding proteins and many proteins including midzone proteins lost their microtubule binding affinity by the inhibition of Aurora-B kinase 6. Thus Aurora-B kinase dependent phosphorylation positively regulates microtubule binding of these proteins but their phosphorylation sites remain to be determined.

In this study, we took a chemical approach using small molecule kinase inhibitors, combined with SILAC (Stable Isotope Labeling with Amino acids in Cell culture) based quantitative proteomics to identify potential substrates of Aurora B kinase during the C-phase. To inhibit Aurora-B kinase two small molecule inhibitors were used VX680 and AZD1152. VX680 is a potent and selective inhibitor of Aurora kinases with inhibition constant values of Aurora-A ~0.6nM, Aurora-B ~18nM, Aurora-C ~4.6nM. 7. On the other hand AZD1152 is a highly potent and selective for Aurora B kinase with inhibition constant values of Aurora A ~1.36 nmol/L , Aurora B 0.37 nmol/L and Aurora C 17 nmol/L 8.



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Several large-scale proteomic studies have covered a significant number of phosphorylation sites during mitosis 9. Using small molecule inhibitors of Aurora-A, Aurora-B and Plk1, phosphoproteomic studies identified phosphorylation sites that are substrates of these enzymes in mitotic spindle and cytoplasm of mitotic cells

10

.

Both Aurora kinase and Polo kinase remain active during cytokinesis but identifying their substrates in large-scale phosphoproteomic analysis is yet to be determined. In this study, we took advantage of monopolar cytokinesis to identify substrates of Aurora-B kinase during the C-phase. Using the quantitative proteomic approach, SILAC, we analyzed the phosphoproteome of purvalanol-A induced C phase upon VX680 mediated Aurora kinase inhibition. Our analysis quantified over twenty thousand phosphopeptides in response to the Aurora-B kinase inhibition; 246 unique phosphopeptides were significantly down-regulated and 74 were up-regulated. Analysis of the down-regulated sites containing proteins suggested that the main targets of Aurora B during cytokinesis are cytoskeletal proteins particularly intermediate filaments.

MATERIALS AND METHODS

CELL GROWTH AND ARRESTS: HeLa S3 cells were grown in Dulbecco’s Modified Eagle’s Medium (Caisson Laboratories) without Lysine and Arginine with 10% dialyzed fetal bovine serum (Sigma, F0392). Either light 12C and 14N Lys(0) and Arg(0) (Sigma) aminoacids were added in 0.146 g/L and 0.08 4g/L concentration respectively which is referred to as “light” media. Or heavy 13C and 15N Lys(8) and Arg(10) (Sigma) aminoacids


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were added in the same concentration to the media referred to as “heavy” media. One cell population was grown in a medium containing heavy lysine and arginine (15N and 13C labeled) for several generations, and a parallel culture in light medium. Initially cells were arrested at mitosis by treating them with 2 mM Thymidine (Sigma) for 16 hours; subsequently, cells were treated with 10µM STC (S-L trityl-L-cysteine) for 12 hours. To induce the C-phase, light cells were treated with 100 µM of Purvalanol A (Tocris Bioscience) for 15 min and heavy cells treated with purvalanolA and 400nM VX680 simultaneously. Cells were washed extensively with PBS, and frozen in liquid nitrogen. C-phase cells VX680-(light) and VX680+ (heavy) cells were also prepared in the same way. Subsequently, immunostaining was performed to confirm the VX680 effect. Similarly for the AZD1152 treatment, light labeled cells were treated with 100 µM of Purvalanol A (Tocris Bioscience, 1580) for 15 min and heavy cells treated with purvalanol-A and 1 µM AZD115 simultaneously to induce their entrance to cytokinesis. C-phase cells AZD115- (light) and AZD115+ (heavy) cells were washed extensively with PBS and harvested.

IMMUNOFLORESENCE VIM-LAP expression Hela cells were fixed in 4% Paraformaldehyde for 15 minutes at room temparature. Cells were blocked with 2 % BSA in PBS-0.1% Triton-X for 30 minutes. Cells were incubated with primary antibodies in 2 % BSA in PBS overnight at 4oC, washed, and then incubated with secondary antibodies and DAPI. Finally, coverslips were mounted on mounting medium (Sigma-Aldrich, M1289) and sealed. Imaging was performed using Nikon 90i confocal microscopy.

ANTIBODIES:



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Immunostaining and western blotting was performed as explained in 6. The following primary antibodies were used: PRC1 (sc-8356), (DM1A) (Sigma-Aldrich), Phospho Histone H3 (06-570; Upstate), alpha-tubulin(3873S; Cell Signaling), actin ab6276.

PHOSPHOPEPTIDE ENRICHMENT: VX680 (-/+) C-phase cells were lysed in a buffer (50 mM Hepes, pH 7.4, 150mM KCl, 1 mM MgCl2, 10 % glycerol, 1mM EGTA, 0.5 %NP-40, 1mM DTT) containing protease inhibitor (Roche EDTA-free cocktail tablet) and phosphatase inhibitors (1M Okadaic acid, 1M microcystine, 10mM NaF, 1mM sodium orthovanadate, 1mM -glycerolphosphate, 1mM sodiumpyrophosphate). Lysates were pre-cleared at 14,000 rpm for 5 min. Cell lysates were mixed in an equal concentration using Bradford assay. The mixed samples were run in an SDS PAGE gel and the gel was cut into 10 slices; each gel slice was digested using trypsin. For each fraction, TiO2 based phosphopeptide enrichment protocol was performed 11. Peptides were dissolved in 100 µL binding buffer (80% (vol/vol) acetonitrile and 6% (vol/vol) TFA). Then, the material was equilibrated by adding loading buffer onto the GELoader spin tip TiO2 columns. Then material was washed twice with the same buffer. Then samples were slowly loaded onto the column using a syringe. Subsequently the column was first washed with the washing buffer (50% (vol/vol) acetonitrile and 0.1% (vol/vol) TFA). Phosphopeptides were eluted with 50ul 10% (vol/vol) NH3·H2O, pH 11.0 into the Eppendorf tubes containing 35 µl of 10% (vol/vol) FA. 1/3 of the samples were directly injected to LC-MS/MS12.

AZD1152 (-/+) C-phase cells were lysed in a buffer containing 50 mM ammonium bicarbonate (pH 8.0), 8 M urea, 1 mM sodium orthovanadate, complete EDTA-free


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protease inhibitor mixture (Roche) and phosSTOP phosphatase inhibitor mixture (Roche). Prior to digestion by Sequencing Grade Modified Trypsin (Promega) the buffer was diluted to 1 M urea using 50 mM ammonium bicarbonate. The resulting peptides were extracted and fractionated on an Agilent 3100 OFFGel Fractionator by using 12-cm IPG strips according to the manufacturer's instructions. Subsequently phosphopeptide enrichment protocol was followed.

DATA ACQUISITION:

Peptides derived from the phosphopeptide enrichment procedure were analyzed by online online C18 nanoflow reversed-phase nLC (Dionex, Thermo Scientific) combined with orbitrap mass spectrometer (Q Exactive Orbitrap, Thermo Scientific) or C18 nanoflow reversed-phase HPLC (Eksigent nanoLC•2D™) combined with a linear ion trap/orbitrap mass spectrometer (LTQ-Orbitrap, Thermo Scientific). Samples were loaded onto an in-house packed 100 µm i.d. × 15 cm C18 column 100 µm i.d. × 17 cm C18 column (Reprosil-Gold C18, 5 µm, 200Å, Dr. Maisch) and separated at 300 nL/min with 90 min linear gradients from 5 to 30% acetonitrile in 0.1% formic acid. The scan sequence began with an MS1 spectrum (Orbitrap analysis; resolution 70,000; mass range 400−2000 m/z; automatic gain control (AGC) target 1e6; maximum injection time 250 ms). Up to 10 of the most intense ions per cycle were fragmented and analyzed in the orbitrap. MS2 analysis consisted of collision-induced dissociation (Higher-energy collisional dissociation (HCD)) (Resolution 17,500; AGC 2e5; normalized collision energy (NCE) 35; maximum injection time 120 ms; charge exclusion unasssigned, 1, 7, 8, >8; dynamic exclusion 30,0 s). The isolation window for MS/MS was 2,0 m/z.



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DATABASE SEARCHES: Raw files were processed using version 1.5.2.8 of MaxQuant

13

(The motif analysis

was supported by 1.0.13.5 version of MaxQuant 13). Cysteine carbamidomethylation was used as a fixed modification, and oxidation (M), deamidation (N), N-acetyl (protein N-term), Phospho (ST) and Phospho (Y) were set as variable modifications. Two missed tryptic cleavages were allowed and the minimal length required for a peptide was six amino acids. The initial precursor mass tolerance was set to 20 ppm and the fragment mass tolerance was set to 0.5 Da. The peptide false discovery rates (FDR) were set to 0.01 and protein (FDR) were 0.01, all other parameters were default settings. The data sets were searched against the human Swissprot/Uniprot database (24.06.2014). Labeling on Lysine and Arginine was set to doublets of 0/0 and 8/10. The resulting output tables of MaxQuant analysis include phosphorylation site probabilities 9c, 13.

MASS

SPECTROMETRIC

SRM

ASSAY

DEVELOPMENT

WITH

SYNTHETIC PEPTIDE STANDARDS The synthetic phosphorylated and non-phosphorylated peptides were supplied by JPT Peptide Technologies. The concentration of each peptide was adjusted to 50fmol/uL. The transition lists were created in Skyline v3.1 software (MacCoss Lab). Primarily, high numbers of transitions, all possible y-ion and b-ion series that matches the criteria (from m/z > precursor-6 to last ion-2 precursor), were selected for each tryptic peptide maximum 2 miss-cleavage, at both 2+ and 3+ charge states. The peptide mixtures were analyzed by nano LC−MS/MS using a TSQ Vantage triple quadrupole mass spectrometer equipped with an Easy n-LC II (Thermo Scientific).


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The samples were injected onto an Easy Spray PepMap column (Thermo Scientific), tryptic peptides were separated on a 75 µm × 150 mm fused silica column packed with ReproSil C18 (3 µm, 100 Å).

SRM assay was developed with Skyline based on synthetic peptide fragmentation. Analysis of samples and the synthetic peptides were performed with a 70 minutes linear gradient from 3% to 35% in 60 minutes, acetonitrile containing 0.1% formic acid, at the flow rate of 300 nL/min. The MS analysis was conducted in positive ion mode with the spray voltage set to 2400 V. The transfer capillary temperature was set to 320 °C, and tuned S lens value was used. SRM transitions were acquired in Q1 and Q3 operated at unit resolution (0.7 fwhm), and the collision gas pressure in Q2 was set to 1.5 mTorr. The cycle time was 3 s in the method. The best transitions (6-8 per precursor) were selected by manual inspection of the data in Skyline, and scheduled transition lists were created for the final assays. Collision energies were optimized for each transition and used accordingly.

PULLDOWN using GFP-TRAP AND PROTEOMIC ANALYSIS Vimentin LAP expressing Hela cells was a gift from Anthony Hyman MPI-CBG, Dresden

14

. Cell pellet was lysed in a buffer (20mM Tris-CL pH7.4, 150mM NaCl,

1mM MgCl2, 10%Glycerol, 0.5mM EDTA, 10mM NaF, 0.5%NP-40, 1mM DTT, EDTA free protease inhibitor (Pierce) and phosSTOP phosphatase inhibitor mixture (Roche). The cleared cell lysate was incubated with GFP-Trap®_A beads (Chromotek). After the washing steps beads were re-suspended in l 2X Laemmli Sample Buffer (4% (w/v) SDS, 20% Glycerol, 120mM Tris-Cl (pH 6.8), 0.02% (w/v) bromophenol blue, 100mM DTT) and boiled to dissociate the immuno-complexes.



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The elutes were run on the SDS-PAGE and the bands corresponding to the VIM::GFP were processed and analyzed in the mass spectrometry as explained above. ProteinPilot was used to analyze the data with the following parameters: 95% or higher protein detection confidence, phosphorylation emphasis using Homo sapiens, Uniprot database 15. The intensity of phosphopeptide, containing the phosphosites S55 or S56, was normalized according to relative ratios of sum nonphosphopeptide intensities for vimentin.

STATISTICAL ANALYSIS:

The statistical analysis of the phosphopeptides was done in three steps: First, data aggregation and intensity normalization was performed. Second, a threshold for deciding whether a peptide is up - or down-regulated was estimated. Third, the phosphopeptide identifications from multiple acquisitions were combined and tested for statistical significance. The analysis was based on the MaxQuant results. Before processing the files peptide ratios were logarithmized and hits to the decoy database and identifications by site were removed from the data. The experimentsal set-up as well as an example of the described analysis is available as Supplementary Figure 1.

Normalization:

Before the significance analysis was done results from technical replicates were aggregated. If a peptide was quantified multiple times over the technical replicates (of one biological replicate) the peptide ratios were determined by taking the mean over all obtained peptide ratios. Thereby, consensus peptide ratios for a biological replicate were obtained and the effect of random precursor selection for MS2 was reduced. Note that two peptides with the same sequence but different phosphorylation sites are


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treated as separate peptides. The aggregated results were then normalized to reduce systematic bias. All peptide ratios were transformed such that the median value of all non-phosphorylated peptides is zero. This was achieved by calculating the median of the peptide ratios and computing a correction factor as the difference between the median and zero. Accordingly, all ratios – including the ratios for the phosphopeptides - were shifted by the derived correction factor.

Threshold estimation:

Instead of using a fixed fold change threshold (e.g. |2|) as cut-off for significant up- or down regulated phosphopeptides we followed a different approach that takes the expression changes of non-phosphorylated peptides as a reference. The threshold was derived by fitting a normal distribution, (, ) to the logarithmic fold changes of the non-phosphorylated peptide ratios. The mean, µ and the standard deviation, σ were estimated from the sample median and the sample median absolute deviation (mad) as more robust estimators for µ and σ, respectively (reduces the influences of outliers). The derived normal distribution fit was then used to derive ‘confidence intervals’ based on the corresponding percentiles of the significance value α which was set to 0.01. The significant thresholds then correspond to the 0.005 percentile and the 0.995 percentile of the fitted normal distribution. For = 0.01 the thresholds were set to significance threshold = ±0.41 (VX data) and = ± 0.31 (AZD data), respectively. All peptides exceeding the respective threshold were regarded as candidates for up-and down-regulation (VX and AZD data were treated separately).

Significant Analysis:



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In the following we describe a method to assign confidence values to expression changes over multiple experiments which was adapted from6. The aim was to decide for each phosphopeptide species whether a significant change in the phosphorylation event is present or not. When data from biological replicates is combined the experiment can be modelled as a binomial distribution 6: If a phosphopeptide was identified in a biological replicate it can either be categorized as significantly regulated or not.

Each biological replicate was treated as independent repeat of the experiment. A phosphopeptide is defined as significantly regulated (up –or down) if its mean ratio (light vs. heavy) exceeds the significance threshold. The number of significantly up-or down-regulated phosphopeptides of the same species (denoted as ) is bounded by the number of times it was observed over all biological replicates (denoted as ). For example, for a peptide that was quantified 5 times out of 6 biological replicates equals 5. If in 2 of the 5 cases the peptide ratio exceeds the significant threshold equals 2.

By

definition,

the

p-value

is

then

calculated

as:

(1 − ) , where equals 0.01 which is the probability of a peptide

exceeding the significance threshold ( ). Finally, the obtained p-values were adjusted for multiple testing by Benjamini-Hochberg correction.

The obtained phosphopeptide results were further annotated by motif annotations available in the Phosphosite database

16

. Further, previously proposed consensus

motifs for the Aurora kinases 17 were used to predict direct targets of the kinase.


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Tests for enrichment of specific cellular components were conducted using the PANTHER classification system

18

; focusing on cellular components (GO-Slim

category)



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RESULTS and DISCUSSION

VX680 treatment of monopolar cytokinesis in Hela Cells

To study the cytokinesis cell cycle stage, followed an established monopolar cytokinesis synchronization protocol as previously described 6. Based on this protocol, the C-phase is induced by treating the S-trityl-L-cysteine (a kinesin-5 inhibitor)- arrested monopolar mitotic cells with the CDK1 inhibitor, Purvalanol-A, for 15-20 minutes. In this setup, to examine the roles of Aurora-B kinase during the C-phase, we used potent inhibitors of Aurora-B, VX680

7, 19

and AZD1152 8. To

avoid complications induced by Aurora-B kinase inhibitor dependent mitotic exit, cells were treated with VX680 or AZD1152 and Purvalanol A simultaneously 5. Purvalanol induced mitotic exit and inactivation of Aurora B kinase activity was confirmed by western blotting using phospho-Histone H3 (S10) antibody VX680

(Figure

1A,

top)

and

AZD1152

(Figure

1A,

20

both for

bottom).

Next,

immunoflourescence was used to examine VX680 treated monopolar cytokinesis cells, mock treated monopolar cytokinesis cells was used as a control. Cells were stained against a midzone protein, PRC1, and microtubules. As previously reported 5, inhibition of Aurora-B kinase blocked cell polarization and midbody assembly during monopolar cytokinesis (Figure 1B).

Aurora-B kinase dependent protein phosphorylation during C-phase

To identify targets of Aurora-B kinase during C-phase, we initially performed phosphopeptide enrichment of monopolar cytokinesis cells either treated with VX680


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or without VX680. In order to compare phosphorylation events quantitatively, we used SILAC. The experimental workflow is summarized in Figure 1C. Heavy cells were arrested in monopolar cytokinesis and treated with VX680 whereas light cells were arrested in monopolar cytokinesis without VX680. We combined clarified lysates from +/- VX680 treatment in equal protein amounts. To avoid any possible heavy isotope incorporation bias, we reversed the labels in another biological repeat (Heavy Mock: Light VX680); subsequently, peptide ratios were reversed and combined altogether. The clarified whole cell lysate was fractionated and phosphopeptide enrichment was performed as described in the Material and Methods section. Phosphopeptide identification and quantification were performed using MaxQuant 13.

In total we identified over ten thousand phosphopeptides (10,590) with 1% FDR (False Discovery Rate) using MaxQuant, 5000 of those were unique phosphopeptides. In all the data, only 105 tyrosine phoshorylation were identified, the rest was serine and/or threonine phosphopeptides. Since our phospho data was result of multiple biological and technical repeats as listed in Supplementary Table 1, the same phosphorylation sites was quantified multiple times, this allowed us to perform a robust statistical test to determine the significantly changed phosphorylation sites as described in the Materials and Methods section and Supplementary Table 1. The phosphopeptides significantly changed, up or down regulated upon the VX680 treatment are listed in a separate table Supplementary Table 1A. All identified phosphopeptides are listed in Supplementary Table 1B. In a previous study mitotic phosphorylation sites were mapped to Aurora and Plk kinases using small molecule inhibitors

10a

, Supplementary Table 1C lists the overlap of down regulated



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phosphorylation sites that were identified both in our cytokinesis analysis in this study 10a

.

Figure 2A shows the distribution of peptides (green) and phosphopeptides (blue) based on their fold change in VX680+/- condition. Kinase inhibitor treatment caused the distribution of phosphopeptides to be skewed towards lower ratios than the other peptides. In order to eliminate bias introduced by mixing two cell lysates, all peptides ratios were normalized such that the median of all non-phosphorylated peptides is zero (Figure 2B). Figure 2C shows the distribution of all peptides and phosphopeptides, in a box plot format, before and after normalization. The red lines indicate the thresholds of up and down regulated phosphopeptides as calculated in the Material and Methods section. As expected: There is a wider range of ratios on the distribution of phosphopeptides in comparison to all peptides and outliers of phosphopeptides are more clustered towards down regulation than up-regulation. Our statistical

analysis

identified

151

unique

phosphopeptides

(with

multiple

measurements) as down regulated and 28 unique phosphorylation sites (with multiple measurements) as up regulated (Supplementary Table 1A).

Next, we took an SRM-based assay in order to validate our large-scale phosphoproteome analysis. For this purpose, three down-regulated phophopeptides were chosen, their quantification values are indicated in Figure 3A. For each phosphopeptide, SRM assay parameters were optimized using its synthetic phosphopeptide and Skyline. The synthetic phosphopeptides' transitions are shown at the top of the each box (Figure 3B). The two graphs shown below are the transitions from mock treated (VX680-) and drug treated (VX680+) samples. In agreement with


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the large-scale data, the phosphopeptide transitions are readily measured in (VX680-) cells whereas they are not detectable in (VX680+) cells (Figure 3B).

VX680 is a pan inhibitor of Aurora kinases, where it inhibits both Aurora A and Aurora B. Although it has the highest selectivity for Aurora A7 in our experimental set-up where we analyze the phosphorylation events during cytokinesis, we expected that the down-regulated phosphorylation sites would be more phosphorylated by Aurora-B kinase than Aurora-A. Because during anaphase and cytokinesis Aurora-B is an active kinase

21

whereas Aurora-A activates earlier events of mitosis and gets

degraded by APC/Cdh1 during mitotic exit 22 therefore Aurora A is not active during cytokinesis. Next, we expanded our analysis into another Aurora kinase inhibitor, AZD1152. AZD1152 is a highly selective inhibitor for Aurora B (Ki 0.37 nmol/L) compared with Aurora A (1.36 nmol/L). The normalization of phosphopeptides was performed as in the VX680 treatment; their peptide and phosphopeptide distribution is shown in Supplementary Figure 2. In total 10650 unique phosphopeptide was identified, based on our statistical analysis 101 were down regulated and 50 were upregulated. The phosphopeptides significantly changed, up or down regulated upon the AZD1152 treatment are listed in a separate table Supplementary Table 2A. All identified phosphopeptides are listed in Supplementary Table 2B. The overlap of down regulated phosphorylation sites that were identified both in our cytokinesis analysis in 10a are listed in Supplementary Table 2C.

In total, 13000 phosphopeptides were identified in the cytokinesis cells and 2460 of those were common in both VX680 and AZD1152 dataset (Supplementary Figure



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Page 20 of 43

3). Next, we compared the down-regulated phosphopeptides upon VX680 and AZD1152 mediated inhibition. In total 236 phosphopeptides were down regulated, 16 of them were common in VX680 and AZD1152 treatment (Figure 4A, left). 101 of the down-regulated phosphopeptides carry Aurora consensus motif and 10 of those are common for both VX680 and AZD1152 treatment (Figure 4A, right). Motif analysis revealed canonical kinase motifs for both down and up regulated phosphorylation sites. As expected, all aurora sites were down regulated in both VX680 and AZD1152 data set (Figure 4B). Multiple kinases implicated in cytokinesis cross talk with aurora kinase in both VX680 and AZD1152 treated cells such as Plk1, Plk3 23, Nek6 24, ROCK1 25. In a previous phosphoproteome analysis, a functional interaction between Plk1 and Aurora A was observed

10b

. In this analysis

we observed that during the C-phase, Aurora kinase inhibition caused down regulation of polo box domain containing phosphopeptides in both VX680 and AZD1152 treated cell. On the other hand, PLK1 and PLK3 motif containing phoshopeptides were also up regulated upon aurora inhibition. Casein kinase 1 (CK1) and casein kinase (CK2) motif containing phosphopeptides were dramatically down regulated upon aurora inhibition (Figure 4B). Both kinases are implicated in the cell division; a recent study in yeast cells showed that overlapping targets of CK2 and Aurora B kinase drives faithful chromosome segregation during anaphase. Our analysis suggests the conservation of the CK2 and Aurora cooperation in mammalian cell division. Further study is required to investigate the role and the molecular mechanism of cross talk between Aurora kinase and other cytokinesis related kinases.

To identify numerically dominant consensus phosphopeptide sequences of significantly changed phosphopeptides, we used weblogo 3.3


26

.For aurora sites

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which exhibit the canonical (R/K)X(S/T)) motif

10b, 27

, we observed that there is a

good agreement in between VX680 (Figure 4C, right) and AZD1152 (Figure 4C, left) treatment. Detailed features of the dominant motif from both treatments are more reminiscent of previously reported Aurora-B sites than Aurora-A sites by having arginine in the position - 2 and -1

10b

. However there are also differences in the

identified motif and previously reported variations of Aurora-B dominant motifs. Perhaps the discrepancy is partly due to different cell cycle stages. In the previous study, the Aurora kinase inhibitors were treated at metaphase 10b whereas in our study the cells were treated at the C-phase. The dominant consensus of the unknown motif was RR(S)P/L was not assigned to any kinase family but consistent in both VX680 and AZD1152 treatments.

Targets of Aurora kinase during cytokinesis

In order to decipher the targets of Aurora B kinase during cytokinesis, we focused on the down-regulated phosphorylation sites of VX680 and AZD1152 treatments. GO annotation analysis of all down regulated proteins reveals that the most significantly enriched categories from both VX680 and AZD1152 treatment are intermediate filament cytoskeleton and in general cytoskeletal elements including microtubule and actin cytoskeleton (Figure 5A).

Previously we showed that during the C-phase, most cytoskeleton proteins lost their affinity to the microtubules upon VX680-treatment. Therefore, both studies support the conclusion that Aurora-B kinase broadly regulates the C-phase cytoskeleton. Other notable enriched categories are chromosome, cell cycle related and plasma



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membrane proteins whose are all actively modulated during cytokinesis (Figure 5B). On the other hand, down regulated proteins with an enzyme activity involve two small GTPase regulators arhgef2 and racgap1 (MgcRacGap) 28. Racgap1 is a subunit of the centralspindlin protein complex, which has a well-established role during cytokinesis. Previously it was shown that MgcRacGap is a substrate of Aurora B kinase during cytokinesis and phoshorylation of MgcRacGap at Ser387 has been suggested to convert its GAP activity from RacGap to RhoA, which is essential for the completion of cytokinesis 29. In this study we investigated another serine residue, Ser203 that is down regulated upon VX680 treatment. Whether this phosphorylation has any biological significance remains to be investigated. ArhGef2 (GEF-H1) is a guanine nucleotide exchange factor that activates Rho GTPase, which is indispensable for cytokinesis 30. Recently, it has been shown that GEF-H1 dependent Rho activation regulates vesicles trafficking pathways of endocytic recycling and exocytosis 31. The phoshorylation site of ArhGef2 (GEF-H1) that we identified in this study was previously reported as an Aurora A dependent phosphorylation site. In agreement with our study, this phosphorylation site was depleted upon Aurora A-B inhibitor AZ447439 or Hesperadin treatment

30

. In addition, two subunits of PP1/PP2

phosphatases were affected by the Aurora kinase inhibition (Figure 5B). A recent study highlights the importance of cross talks between Aurora B kinase and PP1/PP2A phosphatases to create a feedback control mechanism for coordinating the late mitotic events

32

. Listed targets are not the direct targets of Aurora kinase but

rather in direct. Detailed analysis of any one of these interactions will yield more insights into role of Aurora kinase and protein phosphorylation during the C-phase.

To determine protein-protein interactions of proteins with down regulated


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phoshorylation sites upon VX680 treatment, we analyzed our data using the STRING database (Figure 5C). In the protein interactions of down-regulated proteins, the most notable interactions are between cytoskeleton related proteins including vimentin, anillin, racgap1, ArhGef2 (GEF-H1), numa1, KIF2C, KIF23 and TPX2. Additionally multiple keratin proteins (Keratin8, keratin19, keratin7, keratin17, keratin18) and myosin protein (MYO9B) is another remarkable interaction module (Figure 5C).

Intermediate Filaments are targets of Aurora-B kinase during cytokinesis

Our analysis identified intermediate filaments as one of the main targets of Aurora kinase during cytokinesis. Multiple keratin family members, vimentin and lamin A phosphorylations were down regulated upon both VX680 and AZD1152 treatment. Vimentin is a known substrate of Aurora B kinase. Previous studies showed that Aurora B kinase phosphorylates vimentin S72 at the cleavage furrow and vimetin phosphorylation at the cleavage furrow by Aurora-B kinase controls vimentin filaments segregation 33. We imaged Vimentin-::GFP expressing cells upon AZD1152 or VX680 treatment and as reported disorganization of vimentin filaments are observed in response to the Aurora B kinase inhibition during cytokinesis (Figure 6A). In order further study the phosphorylation pattern of vimentin protein in control versus AZD1152/VX680 treatment, Vimentin::GFP protein was pulled down using GFP-trap protein (Figure 6B). Isolated Vimentin::GFP protein was analyzed in the mass spectrometry. The peptide coverage of the vimentin protein was to similar extent from control, VX680 and AZD1152 treated cells (85, 83, 81% respectively). According to the analysis, vimentin S55 and S56 phosphorylations are dimished upon both VX680 and AZD1152 treatment (Figure 6C). These phosphorylation sites were



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also identified in the global analysis as significantly reduced phosphorylation sites (Supplementary Table 2). The function of phosphorylation of vimentin S55 and S56 during cytokinesis remains to be determined. List of all identified peptides of vimentin are listed in Supplementary Table3.

In summary, inhibition of Aurora B kinase dramatically impairs the organization of monopolar cytokinesis. Phosphoproteomic analysis of Aurora kinase inhibition showed that Aurora kinase targets a wide range of proteins in monopolar cytokinesis including cytoskeletal and membrane proteins. These data represent a valuable resource to elucidate how particular proteins are regulated in the C-phase. Further analysis of Aurora dependent phosphorylation sites will provide a better understanding of how Aurora B regulates regional organization of cytoskeleton during cytokinesis and will yield more insights into local biochemistry of other cytokinesis and late mitotic events. An Aurora B kinase dependent phosphorylation site on Histone H3 protein serves as a standard substrate for mitosis 20. In this study we quantified over 12,000 phosphorylation sites and a robust statistical analysis identified a small set of phosphorylation sites those are phosphorylated in Aurora Kinase dependent manner during cytokinesis. The phosphorylation sites identified in this study may have value to serve as a late mitosis and cytokinesis marker.


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Acknowledgements

The authors would like to thank Victoria Taylor, Koc University, for the editing in the manuscript and Büşra Akarlar for the technical support. N. Ozlu is funded by EMBO (European Molecular Biology Organization) Installation Grant, European Union Marie Curie Career Integration Grant and AstraZeneca Open Innovation Grant. BYR gratefully acknowledges funding from Deutsche Forschungsgemeinschaft (DFG), grant number RE3474/2-1.

Supporting Information Available: This material is available free of charge via the Internet at http://pubs.acs.org.



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REFERENCES

1.

Green, R. A.; Paluch, E.; Oegema, K., Cytokinesis in animal cells. Annu Rev

Cell Dev Biol 2012, 28, 29-58. 2.

Canman, J. C.; Hoffman, D. B.; Salmon, E. D., The role of pre- and post-

anaphase microtubules in the cytokinesis phase of the cell cycle. Curr Biol 2000, 10, 611-4. 3.

Glotzer, M., The molecular requirements for cytokinesis. Science 2005,

307 , 1735-9. 4.

Eggert, U. S.; Mitchison, T. J.; Field, C. M., Animal cytokinesis: from parts

list to mechanisms. Annu Rev Biochem 2006, 75, 543-66. 5.

Hu, C. K.; Coughlin, M.; Field, C. M.; Mitchison, T. J., Cell polarization during

monopolar cytokinesis. J Cell Biol 2008, 181, 195-202. 6.

Ozlu, N.; Monigatti, F.; Renard, B. Y.; Field, C. M.; Steen, H.; Mitchison, T. J.;

Steen, J. J., Binding partner switching on microtubules and aurora-B in the mitosis to cytokinesis transition. Mol Cell Proteomics 2010, 9, 336-50. 7.

Harrington, E. A.; Bebbington, D.; Moore, J.; Rasmussen, R. K.; Ajose-

Adeogun, A. O.; Nakayama, T.; Graham, J. A.; Demur, C.; Hercend, T.; Diu-Hercend, A.; Su, M.; Golec, J. M.; Miller, K. M., VX-680, a potent and selective small-molecule inhibitor of the Aurora kinases, suppresses tumor growth in vivo. Nat Med 2004, 10 , 262-7. 8.

(a) Yang, J.; Ikezoe, T.; Nishioka, C.; Tasaka, T.; Taniguchi, A.; Kuwayama,

Y.; Komatsu, N.; Bandobashi, K.; Togitani, K.; Koeffler, H. P.; Taguchi, H.; Yokoyama, A., AZD1152, a novel and selective aurora B kinase inhibitor, induces growth arrest, apoptosis, and sensitization for tubulin depolymerizing agent or


Page 26 of 43

Page 27 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


topoisomerase II inhibitor in human acute leukemia cells in vitro and in vivo. Blood 2007, 110 , 2034-40; (b) Wilkinson, R. W.; Odedra, R.; Heaton, S. P.; Wedge, S. R.; Keen, N. J.; Crafter, C.; Foster, J. R.; Brady, M. C.; Bigley, A.; Brown, E.; Byth, K. F.; Barrass, N. C.; Mundt, K. E.; Foote, K. M.; Heron, N. M.; Jung, F. H.; Mortlock, A. A.; Boyle, F. T.; Green, S., AZD1152, a selective inhibitor of Aurora B kinase, inhibits human tumor xenograft growth by inducing apoptosis. Clin Cancer Res 2007, 13, 3682-8. 9.

(a) Dephoure, N.; Zhou, C.; Villen, J.; Beausoleil, S. A.; Bakalarski, C. E.;

Elledge, S. J.; Gygi, S. P., A quantitative atlas of mitotic phosphorylation. Proc Natl Acad Sci U S A 2008, 105, 10762-7; (b) Lemeer, S.; Heck, A. J., The phosphoproteomics data explosion. Curr Opin Chem Biol 2009; (c) Olsen, J. V.; Blagoev, B.; Gnad, F.; Macek, B.; Kumar, C.; Mortensen, P.; Mann, M., Global, in vivo, and site-specific phosphorylation dynamics in signaling networks. Cell 2006, 127, 635-48; (d) Olsen, J. V.; Vermeulen, M.; Santamaria, A.; Kumar, C.; Miller, M. L.; Jensen, L. J.; Gnad, F.; Cox, J.; Jensen, T. S.; Nigg, E. A.; Brunak, S.; Mann,

M.,

Quantitative

phosphoproteomics

reveals

widespread

full

phosphorylation site occupancy during mitosis. Sci Signal 2010, 3, ra3; (e) Malik, R.; Lenobel, R.; Santamaria, A.; Ries, A.; Nigg, E. A.; Korner, R., Quantitative analysis of the human spindle phosphoproteome at distinct mitotic stages. J Proteome Res 2009, 8, 4553-63. 10.

(a) Kettenbach, A. N.; Schweppe, D. K.; Faherty, B. K.; Pechenick, D.;

Pletnev, A. A.; Gerber, S. A., Quantitative phosphoproteomics identifies substrates and functional modules of Aurora and Polo-like kinase activities in mitotic cells. Sci Signal 2011, 4, rs5; (b) Santamaria, A.; Wang, B.; Elowe, S.; Malik, R.; Zhang, F.; Bauer, M.; Schmidt, A.; Sillje, H. H.; Korner, R.; Nigg, E. A., The Plk1-dependent



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

phosphoproteome of the early mitotic spindle. Mol Cell Proteomics 2011, 10, M110 004457. 11.

(a) Pinkse, M. W.; Uitto, P. M.; Hilhorst, M. J.; Ooms, B.; Heck, A. J., Selective

isolation at the femtomole level of phosphopeptides from proteolytic digests using 2D-NanoLC-ESI-MS/MS and titanium oxide precolumns. Anal Chem 2004, 76, 3935-43; (b) Larsen, M. R.; Thingholm, T. E.; Jensen, O. N.; Roepstorff, P.; Jorgensen, T. J., Highly selective enrichment of phosphorylated peptides from peptide mixtures using titanium dioxide microcolumns. Mol Cell Proteomics 2005, 4, 873-86. 12.

Ozlu, N.; Kirchner, M.; Steen, J. J., Measuring phosphorylation-specific

changes in response to kinase inhibitors in mammalian cells using quantitative proteomics. Methods Mol Biol 2012, 795, 217-31. 13.

Cox, J.; Matic, I.; Hilger, M.; Nagaraj, N.; Selbach, M.; Olsen, J. V.; Mann, M., A

practical guide to the MaxQuant computational platform for SILAC-based quantitative proteomics. Nat Protoc 2009, 4, 698-705. 14.

Poser, I.; Sarov, M.; Hutchins, J. R.; Heriche, J. K.; Toyoda, Y.; Pozniakovsky,

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.; Mechtler, K.; Durbin, R.; Stewart, A. F.; Peters, J. M.; Buchholz, F.; Hyman, A. A., BAC TransgeneOmics: a high-throughput method for exploration of protein function in mammals. Nat Methods 2008, 5, 409-15. 15.

Shilov, I. V.; Seymour, S. L.; Patel, A. A.; Loboda, A.; Tang, W. H.; Keating, S.

P.; Hunter, C. L.; Nuwaysir, L. M.; Schaeffer, D. A., The Paragon Algorithm, a next generation search engine that uses sequence temperature values and feature


Page 28 of 43

Page 29 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


probabilities to identify peptides from tandem mass spectra. Mol Cell Proteomics 2007, 6, 1638-55. 16.

Hornbeck, P. V.; Chabra, I.; Kornhauser, J. M.; Skrzypek, E.; Zhang, B.,

PhosphoSite: A bioinformatics resource dedicated to physiological protein phosphorylation. Proteomics 2004, 4, 1551-61. 17.

(a) Ferrari, S.; Marin, O.; Pagano, M. A.; Meggio, F.; Hess, D.; El-Shemerly,

M.; Krystyniak, A.; Pinna, L. A., Aurora-A site specificity: a study with synthetic peptide substrates. Biochem J 2005, 390, 293-302; (b) Ohashi, S.; Sakashita, G.; Ban, R.; Nagasawa, M.; Matsuzaki, H.; Murata, Y.; Taniguchi, H.; Shima, H.; Furukawa, K.; Urano, T., Phospho-regulation of human protein kinase Aurora-A: analysis using anti-phospho-Thr288 monoclonal antibodies. Oncogene 2006, 25, 7691-702; (c) Sardon, T.; Pache, R. A.; Stein, A.; Molina, H.; Vernos, I.; Aloy, P., Uncovering new substrates for Aurora A kinase. EMBO Rep 2010, 11, 977-84. 18.

Mi, H.; Muruganujan, A.; Thomas, P. D., PANTHER in 2013: modeling the

evolution of gene function, and other gene attributes, in the context of phylogenetic trees. Nucleic Acids Res 2013, 41, D377-86. 19.

Taylor, S.; Peters, J. M., Polo and Aurora kinases: lessons derived from

chemical biology. Curr Opin Cell Biol 2008, 20, 77-84. 20.

Hirota, T.; Lipp, J. J.; Toh, B. H.; Peters, J. M., Histone H3 serine 10

phosphorylation by Aurora B causes HP1 dissociation from heterochromatin. Nature 2005, 438, 1176-80. 21.

Nigg, E. A., Mitotic kinases as regulators of cell division and its

checkpoints. Nat Rev Mol Cell Biol 2001, 2, 21-32. 22.

(a) Littlepage, L. E.; Ruderman, J. V., Identification of a new APC/C

recognition domain, the A box, which is required for the Cdh1-dependent



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

destruction of the kinase Aurora-A during mitotic exit. Genes Dev 2002, 16, 2274-85; (b) Crane, R.; Gadea, B.; Littlepage, L.; Wu, H.; Ruderman, J. V., Aurora A, meiosis and mitosis. Biol Cell 2004, 96, 215-29. 23.

Archambault, V.; Lepine, G.; Kachaner, D., Understanding the Polo Kinase

machine. Oncogene 2015. 24.

Fry, A. M.; O'Regan, L.; Sabir, S. R.; Bayliss, R., Cell cycle regulation by the

NEK family of protein kinases. J Cell Sci 2012, 125, 4423-33. 25.

Thumkeo, D.; Watanabe, S.; Narumiya, S., Physiological roles of Rho and

Rho effectors in mammals. Eur J Cell Biol 2013, 92, 303-15. 26.

Crooks, G. E.; Hon, G.; Chandonia, J. M.; Brenner, S. E., WebLogo: a

sequence logo generator. Genome Res 2004, 14, 1188-90. 27.

Alexander, J.; Lim, D.; Joughin, B. A.; Hegemann, B.; Hutchins, J. R.;

Ehrenberger, T.; Ivins, F.; Sessa, F.; Hudecz, O.; Nigg, E. A.; Fry, A. M.; Musacchio, A.; Stukenberg, P. T.; Mechtler, K.; Peters, J. M.; Smerdon, S. J.; Yaffe, M. B., Spatial exclusivity combined with positive and negative selection of phosphorylation motifs is the basis for context-dependent mitotic signaling. Sci Signal 2011, 4, ra42. 28.

Hall, A., Rho family GTPases. Biochem Soc Trans 2012, 40, 1378-82.

29.

Minoshima, Y.; Kawashima, T.; Hirose, K.; Tonozuka, Y.; Kawajiri, A.; Bao,

Y. C.; Deng, X.; Tatsuka, M.; Narumiya, S.; May, W. S., Jr.; Nosaka, T.; Semba, K.; Inoue, T.; Satoh, T.; Inagaki, M.; Kitamura, T., Phosphorylation by aurora B converts MgcRacGAP to a RhoGAP during cytokinesis. Dev Cell 2003, 4, 549-60.


Page 30 of 43

Page 31 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


30.

Birkenfeld, J.; Nalbant, P.; Bohl, B. P.; Pertz, O.; Hahn, K. M.; Bokoch, G. M.,

GEF-H1 modulates localized RhoA activation during cytokinesis under the control of mitotic kinases. Dev Cell 2007, 12, 699-712. 31.

Pathak, R.; Delorme-Walker, V. D.; Howell, M. C.; Anselmo, A. N.; White, M.

A.; Bokoch, G. M.; Dermardirossian, C., The microtubule-associated Rho activating factor GEF-H1 interacts with exocyst complex to regulate vesicle traffic. Dev Cell l 2012, 23, 397-411. 32.

Afonso, O.; Matos, I.; Pereira, A. J.; Aguiar, P.; Lampson, M. A.; Maiato, H.,

Feedback control of chromosome separation by a midzone Aurora B gradient. Science 2014, 345, 332-6. 33.

Goto, H.; Yasui, Y.; Kawajiri, A.; Nigg, E. A.; Terada, Y.; Tatsuka, M.; Nagata,

K.; Inagaki, M., Aurora-B regulates the cleavage furrow-specific vimentin phosphorylation in the cytokinetic process. J Biol Chem 2003, 278, 8526-30.



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FIGURE LEGENDS

Figure 1. Inhibition of Aurora kinase, via VX680 treatment, blocked cell polarization of monopolar cytokinesis A. Western blotting against a phosphoserine residue on Histone H3, a known AuroraB phosphorylation site, is clearly visible in the mitotic whole cell lysate, which was significantly diminished in Purvalanol A-induced monopolar cytokinesis (CphaseVX680(-)).The band was not detectable in cells treated with purvalanol-A and VX680 simultaneously treated cell lysates in (Cphase-VX(+)) (bottom). The loading control from the same blot is shown at the top panel, using Tubulin antibodies (Top). The same western blotting using cells treated with AZD1152 instead of VX680 (Bottom).

B. Immunostaining of Purvalanol A-induced monopolar cytokinesis Hela cells were probed for a midzone protein PRC1 (blue) and microtubules (green) after mock (top) or VX680 treatment. Highly polarized microtubules and PRC1 localization at the tip of microtubules (top) were disrupted in VX680 treated cells (bottom). Bar 5 µm. C. The experimental workflow. Green, grey and red colors represent microtubules, chromosomes and a cortical furrow maker, respectively.

Figure 2. VX680 -/+ Phosphorylation Analysis A. Isotope ratio distribution of all peptides versus their intensities. The X axis shows the fold change (fc) on a log2 scale and the Y axis shows the intensities of all peptides on a log scale. Blue represents phoshopeptides and green represents all other identified peptides.


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B. Threshold estimation from non-phosphorylated peptides. The black line shows the normal distribution fit of the log2 fold change values. Red vertical lines indicate the corresponding confidence intervals at =. that are used as the significance threshold. C. Normalization effect on peptides. The plot shows the distribution of log2(fold changes) before and after normalization for both phosphorylated and all peptides.

Figure 3. SRM analysis of the down regulated phophopeptides identified in VX680-/+ A. SRM transition results for the down peptides as shown in the table. B. FSGGFGAR, SIGSAVDQGNESIVAK, SLAALSQIAYQR, SLGSAGPSGTLPR peptides are diminishes after drug treatment. Each box reserved for one peptide. At the top single graph shows the transitions of the synthetic phosphopeptide. The two graphs shown below are the transitions of the phosphopeptide present in the sample. VX680 (+) is used for drug treated sample and VX680 (-) is used for non-treated one.

Figure 4. Analysis of down and up-regulated phosphorylation site motifs

A. Comparison of phospho-peptides that are significantly down regulated upon VX680 (green) or AZD1152 (blue) treatment. Left Venn diagram represents all down-regulated phosphopeptides, the right one only shows the down-regulated phosphopeptides carrying a consensus Aurora motif. B. Discovered motifs of down-regulated (red) and up-regulated (blue) phoshopeptides identified by MaxQuant by treating the cells either with VX680 (left) or AZD1152



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(right). Annotated sites are labeled with their respective motif name and the y-axis shows their frequency. C. The consensus motif of the assigned Aurora motif logo from the down-regulated phosphopeptides either treated with VX680 (left) or AZD1152 (right) inhibitors. D. The motif logo of all peptides with an unknown site (only from down-regulated peptides either treated with VX680 (left) or AZD1152 (right) inhibitors. Consensus was extracted using weblogo 3.4.

Figure 5. Analysis of the proteins containing down-regulated phosphorylation sites upon Aurora kinase inhibition during the C-phase

A. GO annotation (GO-Slim, PANTHER)analysis of proteins with down regulated sites upon Aurora kinase inhibitors (VX680 (green), AZD1152 (blue)) using PANTHER. Bars marked with an ‘*’ represent GO sub-groups that are significantly enriched with p-values below 0.05 (PANTHER overrepresentation test).

B. Down regulated proteins from the listed categorizes based on their cellular component. Percentages reflect the enrichment percentage of each down regulated category. C. Sub-networks of proteins containing down regulated phosphorylation sites using the STRING database; those with Aurora consensus motifs are indicated at the top.

Figure 6. Analysis of Vimentin protein upon Aurora kinase inhibition during cytokinesis


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A. Vimentin::GFP expressing cells treated with none (control, top), AZD1152 (middle) or VX680 (bottom) immunostained: vimentin (green), tubulin (red) and DNA (blue). Upon aurora kinase treatment cytokinesis failed and vimentin protein mislocalized. B. Western blotting analysis GFP pull-down in VIM-LAP expressing cells. Elution from GFP-trap beads and the unbound fractions were blotted against actin and GFP. In addition to VIM::GFP, regular Hela was used as a negative control. VIM::GFP was eluted in similar amount in mock (-), +VX680 and +AZD1152 treated VIM::GFP cells. C. Mass spectrometry analysis of VIM::GFP protein. Vimentin peptide coverage was 85%, 83% and 81% in mock (-), +VX680 and +AZD1152 treated VIM::GFP C-phase cells respectively. In comparison to the normalized sum of nonphospho-vimentin peptide intensity SLYApSpSPGGVYATR peptide intensity was reduced in VX680 and AZD1152 treated C-phase cells. MaxQuant phosphosite probability is SLYApS(0.5)pS(0.5)PGGVYATR.

Supplementary Figure 1. Experimental set-up and statistical analysis: A. VX680 acquisition set-up with exemplary statistical analysis for the fictive peptide VPQPNSAR. Biological replicates (BR1 - BR4) and Rev. BR1 - Rev. BR3 have reversed labels. The number of technical replicates (TR) varies between 1 and 3. Each peptide in the ‘Analysis TR level’ column represents one MS/MS identification. For the analysis on BR level the MS/MS data is first aggregated such that only one averaged (mean) log2 fold change (fc) value per peptide species (per BR) is considered. If that fc exceeds a derived threshold (e.g. 0.41) the phosphopeptide is considered as potentially up- or down-regulated (increases



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

k). If the fc does not exceed the threshold the phosphopeptide is only counted as observed (increases n). At a given significance level (e.g. 0.01) the p-value is then calculated as shown below with k and n as parameters for the binomial. B. AZD1152 acquisition set-up. Two Biological replicates with two technical replicates were acquired for the experiment.

Supplementary Figure 2. AZD1152 -/+ Phosphorylation Analysis A. Isotope ratio distribution of all peptides versus their intensities. The X axis shows the fold change (fc) on a log2 scale and the Y axis shows the intensities of all peptides on a log scale. Blue represents phoshopeptides and green represents all other identified peptides. B. Threshold estimation from non-phosphorylated peptides. The red line shows the normal distribution fit of the log2 fold change values. Black vertical lines indicate the corresponding 99% confidence intervals that is used as the significance threshold. C. Normalization effect on peptides. The plot shows the distribution of log2(fold changes) before and after normalization for both phosphorylated and all peptides.

Supplementary Figure 3. Comparison of phospho-peptides those are identified in VX680 (green) or AZD1152 (blue) data set and their intersection.

Supplementary Table 1. List of all quantified phosphopeptides upon VX680 treatment in monopolar cytokinesis cells A. A list of significantly changed phosphopeptides from SILAC experiments of VX680 and mock treated cells. The listed phosphopeptides are significantly up or


Page 36 of 43

Page 37 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


down regulated upon the VX680 treatment (with p= 0.01 or less). Ratios are listed in log2 scale. B. A list of all phosphopeptides from SILAC experiments of VX680 and mock treated cells. All quantified phosphopeptides in multiple biological repeats with inverted isotopes (Heavy VX680: Light Mock or Heavy Mock: Heavy VX680). C. List of down-regulated phosphopeptides identified in 10a.

Supplementary Table 2. List of all quantified phosphopeptides upon AZD1152 treatment in monopolar cytokinesis cells A. A list of significantly changed phosphopeptides from SILAC experiments of AZD1152 and mock treated cells. The listed phosphopeptides are significantly up or down regulated upon the AZD1152 treatment (with p= 0.01 or less). Ratios are listed in log2 scale. B. A list of all phosphopeptides from SILAC experiments of AZD1152 and mock treated cells. All quantified phosphopeptides in multiple biological repeats with inverted isotopes (Heavy AZD1152: Light Mock). C. List of down-regulated phosphopeptides identified in 10a.

Supplementary Table 3. A list of all peptides (including phosphopeptides) from GFP-TRAP pulldown experiments of VX680/AZD115 treated and control cells. The peptides and phosphopeptides were identified with 95% or higher protein detection confidence using ProteinPilot.


FIGURE 1

B

Cells grown in Lys0, Arg0 Cells grown in Lys8, Arg10

kDa 17

Cphase- AZD1152(+)

Cphase- AZD1152(-)

55

Mitosis

Page 38 of 43

C

Cphase-Vx680(+)

Mitosis

A

1 2 3 4 5 6 7 8 9 10 11 12 pH3 13 14 15 16 Tubulin 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 pH3 35 36 37 38 39 Tubulin 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 Mock 55 56 57 58 59 60

Cphase-Vx680(-)


Thymidine treatment for 16 hours

S-L trityl-L-cysteine (STC) treatment for 12 hours

Purvalanol A

Purvalanol A and VX680 /AZD1152 for 15 minutes

kDa

C-phase + VX680 or AZD1152

C-phase Mock

17

Lyse cells mixed 1:1

55

Tryptic digest

TiO2 phosphopeptide enrichment

PRC1

Microtubules

Merge LC-MS/MS

+VX680 ACS Paragon Plus Environment

APage 39 of 43 10

phosphopeptides peptides

log10 (intensity)

1 2 3 4 5 6 7 8 9 107.5 11 12 13 14 15 16 17 18 19 20 5 21 22 23 24 25 26 3 27 28 29 30 31 32 2 33 34 35 36 37 38 39 1 40 41 42 43 44 45 46 0 47 48 49 50 51 52 2 53 54 55 56 1 57 58 59 60

FIGURE 2


0 log2 (fold change)

-4

Densitiy

B

4

-1.5

log2 (fold change)

C

0 log2 (fold change)

1.5

0

-1

-2


peptides

peptides (normalized)

phospho- phospho-peptides peptides (normalized)

Protein Name Phospho-Peptide CALD1 pSLAALSQIAYQR DDX3X FpSGGFGAR RACGAP1 pSIGSAVDQGNESIVAK y10 - 1120.6109+

700

y9 - 1049.5738+

VX680 +/0,20 0,18 0,21

Direction Down Down Down

n 6 2 4

y8 - 978.5367+

y7 - 865.4526+

k 6 2 4

y6 - 778.4206+

p-value 1E-12 0,0001 0,00000001 b8 - 864.4227+

600 500

pSLAALSQIAYQR

400 300 200 100 0

32.0

4000

32.5

VX680(-)

3000

1200

34.0

VX680(+)

1000

3500

800

2000

600

1500

400

1000

200

500 0

33.5

33.0

Retention Time

2500

32.0

32.5

33.0

y7 - 731.2872+

20000

33.5

y7 -98 - 633.3103+

0 32.0

34.0

y6 - 564.2889+

32.5

y5 - 507.2674+

33.0

34.0

33.5

y3 - 303.1775+

b4 -98 - 331.1401+

FpSGGFGAR

15000

Intensity

1 2 3 4 5 6 B 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

FIGURE Page 3 40 of 43


10000

5000

0

Retention Time

18.5

18.0

VX680(-)

1000

160 140

19.5

19.0

20.0

VX680(+)

120

800

100 600

80

400

60 40

200

20

0

18.0

18.5

y14 - 1374.6859+

19.0

19.5

y13 - 1317.6645+

20.0

y12 - 1230.6325+

0

18.0

y11 - 1159.5953+

2000

18.5

19.0

y10 - 1060.5269+

19.5

b3 -98 - 240.1343+

20.0

b6 -98 - 497.2718+

24.0

pSIGSAVDQGNESIVAK

1500

Intensity

A

1000 500 0 23.0 1600 1400

23.5

Retention Time

VX680(-)

24.0

25.0

24.5 10

VX680(+)

1200 1000 800

5

600 400 200 0

ACS Paragon Plus Environment 23.0

23.5

24.0

24.5

25.0

0

23.0

23.5

24.0

24.5

25.0

Page 41 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 60 20 21 22 23 24 25 40 26 27 28 29 30 20 31 32 33 34 35 36 0 37 38 39 40 41 42 43 4 44 45 46 47 2 48 49 50 0 51 52 53 54 4 55 56 57 58 2 59 60


A

V X 680

A Z D 1152

V X 680

85

52

16

135

Figure 4

A Z D 1152

39

10

B

K S

S

D G E

K

G

P

R S

L

K

D

G

T

P

V E

K

P

A

V A

T

V

10

D

S

S

T

S V

L

L

A G

SA

T

S

P

E

L A Q D

V

N

E

N

V

R V

15 20 VX680 predicted Aurora motif

0

PLK1 PLK3 Polo box ROC K1 ROC K2 PKG 1 iso 2 CK2 A1 AMP KA1

RS

R K S

D

E

R

P R

E

A

L

K

N

D

Q

L Q V

R M

M

ST

V

T

V

F P

LG

G I S L

A L S L G E E P L E D R T EA D

D

L

M P

S

G

E S P V G

K

SV V

A

F

S

G

K

D

R D

K

P

L

T

P

R T

N

T

N

V

10

L K

K

L

P E K A AG R P P S Q G S L

Q

Y

S

15 20 AZD1152 predicted Aurora motif

R T

S

AS SRRK R TA EA S Q T SQ K S

G

L

G Q Q LG V T

N P Y VR V R V A

F

V

P

P A

V

Y

I

Q

V

G

E

H

N

10

TY

A

E

H I H

L L P

M N T

Q N

Y

15 20 VX680 no annotated motifs

R K

E K

KS L

S

0

S

R

E

S

G

G P V G L A I TSV S Q N I A K T K VS R G Q W

W

PSS

RVG TKE

I

K

Q

2

R A PAP EA

G

T

L

D I P F K P E K P E G

bits

bits

4

G G AE

0

P

AT

R

K

S D K

E

G

L F D P P G S T Q R G L Q

Q

V

Y

2

G

LS

I

Q

D

M

4 bits

RS

R

K S

PA KG Q R AG R E R S R L

L

Chk1

PKA PKA /AKT CK1 CK2 AUR ORA AUR ORA -A AurB CAM K2 GSK 3 PKA CA PKC A CDK 1 CHK 1 PKD NEK 6 PAK1 Akt1

bits

C

VX680 up-regulated AZD1152 up-regulated VX680 down-regulated AZD1152 down-regulated

L AG P R P P YV R L R K N E V S D E K

K

E

N

R A

L L G

Q

T

Q

A

S K

T

L P

G I

G

L A G

E S S E L D

G V

T

P P

G

S

Q P D

S P P E

D

G

P

R

A A R L

S

T

T

S

E E T

S

K

L

K

S T

L

A

P

K

P

E

R

F

T

M

Y

10


15 20 AZD1152 no annotated motifs

V

Journal of Proteome Research VX680 down-regulated

** **

* *

2 1

e

ne

mem

ral to

integ

cellular component (GO-Slim PANTHER)

bran

e

mbra

plasm

a me

sm

cytop la

bran

micro tubu le ribon ucleo com prote plex in orga nelle intra cellu lar cell p art macr o o le com mplecular x prote in co mple x actin cytos keleto n

on ribos ome

ojecti

cytos ol

0

cell p r

enrichment

*

3

Figure 5

AZD1152 down-regulated significantly enriched (p < 0.05)

*

4

inter me cytodsiate fila keleto ment n cytos keleto n

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

>5

mem

A

B Chromosome HMGN1 TMPO TOP2A PHF6 POLA1 TPR KIF2C PPP1R12A ZFR SEPT7 TNKS1BP1 FLNA BRD4 HIST1H1C NUMA1 CENPF HNRPK

Plasma Membrane KRT19 ARHGEF2 MAP7 RAB13 EEF1A1 ZYX TNKS1BP1 AHNAK PACSIN3 LMO7

CALD1 TJP2 PLEC PAWR IRS2 SEPT7 FLNA RACGAP1 KRT8 VIM

19.32%

Regulation of Cell Cycle RPS6 MYBBP1A TOP2A TPR PDE3A EIF4EBP1 DDX3X HCFC1 TPX2 BRD4 RACGAP1 RBM14 KIF23 ANLN CENPF

17.05%

22.73%

1.14% 17.05%

Endoplasmic Reticulum 1.14%

2.27%

Protein Kinase Activity SCYL2

RNA Binding MYO18A KRT18 EIF4G1 SCAF4 PABPN1 LRRC47 THRAP3 TPR DDX3X PRRC2C HIST1H1C RBM14

SEC61B SLTM BCLAF1 PLEC MYBBP1A PATL1 TOP2A SRRM2 FLNA DIDO1 NSUN2 HUWE1

5.68%

SEC61B 42.05%

Signal Transducer Activity

VIM LMNB1 ARHGEF2 PAWR KIF2C MYO9B NUMA1

Cytoskeleton CALD1 MAP7 EEF1A1 LASP1 TPX2 HNRPK

MYO18A PLEC TOP2A SEPT7 DIDO1 KRT8

KRT7 STMN1 TPR TNKS1BP1 AHNAK KIF23

KRT19 KRT17 DNM1L FLNA RACGAP1 ANLN

KRT18 LMNA ZYX PPP1R12A NSUN2 CENPF

KRT17 STMN1 IRS2 PPP1R12A FLNA

Phosphotase Activity PPP1R12A PPP2R5D PFKFB2

C


RPS6 MATR3 DDX17 SF1 ZC3HAV1 EEF1A1 PHF6 ZYX ZFR AHNAK HNRPK VIM

Page 42 of 43

A

Page 43 of 43

+ VX680

Figure 6

Vimentin

+ AZD1152

DNA/Tubulin/Vimentin

Intensity

C

HeLa

Vim:GFP AZD1152(+)

Vim:GFP VX680(+)

Vim:GFP (-)

Elute

HeLa

Vim:GFP AZD1152(+)

Unbound

Vim:GFP (-)

HeLa

Cell lysate

Vim:GFP VX680(+)

B

Vim:GFP

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Control

kDa

GFP

81

Actin

42

Vimentin coverages

85%

83%

81%

2,00 E09 1,50 E09

Non phosphopeptide sum intensity

1,00 E09

pSLpYApSpSPGGVYATR

5,00 E08 3,00 E06 2,00 E06 1,00 E06 0,00 E00

Cytokinesis


Cytokinesis VX680

Cytokinesis AZD1152

Phosphoproteomic Analysis of Aurora Kinase Inhibition in Monopolar

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