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Identification of mitosis-specific phosphorylation in mitotic chromosome-associated proteins Shinya Ohta, Michiko Kimura, Shunsuke Takagi, Iyo Toramoto, and Yasushi Ishihama J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.6b00512 • Publication Date (Web): 09 Aug 2016 Downloaded from http://pubs.acs.org on August 10, 2016
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Identification of mitosis-specific phosphorylation in mitotic chromosome-associated proteins
Shinya Ohta1§*, Michiko Kimura2§, Shunsuke Takagi2, Iyo Toramoto1, and Yasushi Ishihama2*
1
Center for Innovative and Translational Medicine
Medical School, Kochi University Kohasu, Oko-cho, Nankoku, Kochi 783-8505, Japan 2
Graduate School of Pharmaceutical Sciences, Kyoto University
46-29 Yoshidashimoadachi-cho, Sakyo-ku, Kyoto 606-8501, Japan
§
These authors equally contributed to this work.
*Co-corresponding authors Shinya Ohta E-mail:
[email protected] Telephone: +81-88-880-2309 Yasushi Ishihama E-mail:
[email protected] Telephone: +81-75-753- 4555 Fax: +81-75-753-4601
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Abstract During mitosis, phosphorylation of chromosome-associated proteins is a key regulatory mechanism. Mass spectrometry has been successfully applied to determine the complete protein composition of mitotic chromosomes, but not to identify post-translational modifications. Here, we quantitatively compared the phosphoproteome of isolated mitotic chromosomes with that of chromosomes in non-synchronized cells. We identified 4,274 total phosphorylation sites and 350 mitosis-specific phosphorylation sites in mitotic chromosome-associated proteins. Significant mitosis-specific phosphorylation in centromere/kinetochore proteins was detected, although the chromosomal association of these proteins did not change throughout the cell cycle. This mitosis-specific phosphorylation might play a key role in regulation of mitosis. Further analysis revealed strong dependency of phosphorylation dynamics on kinase consensus patterns, thus linking the identified phosphorylation sites to known key mitotic kinases. Remarkably, chromosomal axial proteins such as non-SMC subunits of condensin, TopoIIα, and Kif4A, together with the chromosomal periphery protein Ki-67 involved in the establishment of the mitotic chromosomal structure, demonstrated high phosphorylation during mitosis. These findings suggest a novel mechanism for regulation of chromosome restructuring in mitosis via protein phosphorylation. Our study generated a large quantitative database on protein phosphorylation in mitotic and non-mitotic chromosomes, thus providing insights into the dynamics of chromatin protein phosphorylation at mitosis onset. Keywords: Chromosome, Chromatin, Mitosis, and Phosphorylation
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Introduction In eukaryotic cells, protein phosphorylation plays important regulatory roles in chromosome dynamics by causing conformational changes in protein structure, reversibly switching enzyme activities, and determining protein localization and interaction with other molecules.1-3 Thus, cyclin-dependent serine/threonine kinases (CDKs) play a central role in mitosis progression. The phosphorylation activity of CDKs is controlled by the formation of complexes with cyclins, a family of regulatory proteins expressed in different subcellular locations and phases of the cell cycle, which have substrate-binding sites and target CDKs to specific protein substrates. For example, complex formation between M phase-specific cyclin B and CDK1 (cyclin B-CDK1) is necessary for entering mitosis.4 Other well-known mitotic kinases include Aurora B, NIMA-related kinase Nek2, Polo kinase PLK1, and Warts kinase. Mitosis is thus considered to be controlled by hierarchical phosphorylation.5,6 Therefore, to understand the regulatory mechanisms underlying mitosis progression, it is necessary to compare phosphorylation profiles and the activation of specific protein kinases before and during mitosis. During mitosis occurring in higher eukaryotes, chromosomes undergo multiple stages of reorganization: condensation, alignment, segregation, and de-condensation. Despite many decades of investigation, the mechanisms underlying
chromatin
restructuring
(chromosome
condensation
and
de-condensation) in mitosis remain unclear. Points of controversy include the role of non-histone proteins in the regulation of chromosomal structure, and the organization of chromatin fibers in mitosis. As part of a reductionist approach to the analysis of mitotic chromosome structure and function, recent studies have used proteomic methods based on mass spectrometry (MS) to identify chromatin constituents.7-9 A previous study showed that proteins forming the mitotic chromosomal axis played an essential role in the arrangement of structurally stable mitotic chromosomes.10 One of the most important protein complexes in the structure of the chromosomal axis is condensin, which occurs in two forms: 3 ACS Paragon Plus Environment
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condensin I and II.11 These proteins are involved in the assembly of pentameric complexes composed of the SMC2 and SMC4 ATPases and three auxiliary subunits (CapG/G2, CapD2/D3, and CapH/H2). Mitotic kinases such as CDK1 are considered to regulate the two condensin forms in mitosis
4
. Different
phosphorylation-based mechanisms involved in chromosomal restructuring during mitosis have been reported for condensin I and II complexes. Upon phosphorylation by Aurora B, CAP-H, a subunit of condensin I, acquires the ability to interact with histone H2A.Z and to associate with mitotic chromosomes.12 Condensin I gains the ability to introduce positive supercoils to DNA via phosphorylation of another condensin I subunit, CAP-D2, by Cdk1.13,14 In contrast, this supercoiling activity of condensin is negatively regulated by casein kinase 1 (CK1) phosphorylation of CAP-H in Xenopus egg extracts.15 Several unique phosphorylation sites have also been reported in condensin II subunits. Thus, the phosphorylation of condensin II subunit CAP-D3 by Cdk1 and PLK1 is considered to promote proper chromosome condensation in prophase.16 Moreover, it has been shown that the activity of CAP-G2 and CAP-H2 is regulated by phosphorylation.17,18 Another important substructure in mitotic chromosomes modified by phosphorylation is the kinetochore. Phosphorylation by Aurora B regulates the binding of microtubules to kinetochore components at Ser and Thr residues.19-21 Spindle checkpoint is also controlled by protein phosphorylation. Thus, Mad2 phosphorylated by Bub1 kinase is uncoupled from Cdc20, which binds the anaphase-promoting complex and activates its ubiquitin ligase activity; as a result, sister chromatid cohesion is resolved, promoting the metaphase/anaphase transition.22 However, despite the importance of chromosomal restructuring and kinetochore-microtubule attachment in
mitosis,
the
underlying
mechanisms
are
still
unclear.
Unknown
phosphorylation-dependent interactions may be involved in the fine-tuning of chromosomal reorganization in mitosis. A previous study reported whole-cell phosphoproteome profiling in mitosis by MS.23,24 However, whole-cell analysis does not correlate phosphorylation profiles to the functional activity of proteins at the actual reaction locus, i.e., mitotic 4 ACS Paragon Plus Environment
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chromosomes. Therefore, to understand the regulation of chromatin reorganization in mitosis, it is useful to perform a comprehensive analysis of phosphorylation dynamics in the proteins directly associated with chromosomes. Here, we identified 4,274 phosphorylation sites in proteins bound to mitotic chromosomes and quantitatively analyzed phosphorylation at the mitotic entry using SILAC technology. Our study generated a large quantitative dataset on the chromosome-associated phosphoproteome in mitosis, providing insight into phosphorylation dynamics during chromosome segregation.
Experimental Section Cell culture DT40 CDK1AS cells were maintained in RPMI 1640 medium supplemented with 10% (v/v) fetal bovine serum (FBS), 1% chicken serum (CS), 100 U/ml penicillin, 100 µg/ml streptomycin (Wako, Osaka Japan) at 39°C and 5% CO2 in a humid incubator. For labeling lysine and arginine with
13
C and
15
N, cells were
maintained in RPMI without L-Lysine and L-Arginine (Thermo Scientific, Waltham, MA, USA) supplemented with 10% FBS, 100 µg/ml L-Lysine-13C65N2:hydrochloride (K8, Wako) and 30 µg/ml L-Arginine-13C615N4:hydrochloride (R10, Wako), or 100 µg/ml L-Lysine-4,4,5,5-d4:hydrochloride (K4; Sigma-Aldrich, St. Louis, MO, USA) and 30 µg/ml L-Arginine-15N4:hydrochloride (R4; Sigma-Aldrich). Mitotic chromosome isolation DT40
cells
were
incubated
with
4-amino-1-tert-butyl-3-(1′-naphthylmethyl)pyrazolo[3,4-d]pyrimidine
(1-NMPP1;
sc-203214A, Santa Cruz Biotechnology, Dallas, TX, USA) for 20 h, resulting in a mitotic index of > 90%. The cells swollen in hypotonic buffer containing 40 mM KCl for 5 min were disrupted in polyamine-EDTA buffer [0.75 mM Spermidine, 0.3 mM Spermine, 2 mM K-EDTA (pH7.4, Sigma-Aldrich), and 0.1% Digitonin (Biosynth, 5 ACS Paragon Plus Environment
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Staad Switzerland)] using a 15 mL Dounce homogenizer7,25. The mitotic chromosomes were purified using sucrose density gradient (15%, 60%, and 80%) centrifugation followed by Percoll (GE Healthcare Ltd., Buckinghamshire UK) density gradient centrifugation. All buffers used for this mitotic chromosome isolation process contained 1 µg/mL Antipain (Peptide Institute, Inc., Osaka Japan), 1 µg/mL Aprotinin (Wako), 1 µg/mL Chymostatin, 1 µg/mL Leupeptin, 1 µg/mL Pepstatin A (Peptide Institute, Inc.), 0.1 mM phenylmethylsulfonyl fluoride (Wako), and Phosphatase Inhibitor Cocktail 2 and 3 (1:1000, Sigma-Aldrich). Mitotic chromosomes from three independent preparations were pooled together (1.0 ×109 cells; OD260 = 5) and solubilized in phase-transfer surfactant (PTS) buffer.26 Isolation of nuclei Nuclear fractions were prepared as previously described.27 Cells were washed with PBS twice, resuspended in cell lysis buffer [10 mM HEPES; pH 7.9, 10 mM KCl, 0.1 mM EGTA, 0.1 mM EDTA, 1 mM DTT, 0.5 mM PMSF, and phosphatase inhibitor cocktails 2 and 3], and allowed to swell on ice for 15 min; then, NP-40 was added up to 0.5%. Cells were vortexed for 10 s to disrupt cell membranes and then centrifuged at 500 × g for 3 min at 4°C. The pellet containing cell nuclei was washed three times with lysis buffer and resuspended in PTS buffer. Immunoblotting Total protein extracts (10 µg) were resolved by SDS-PAGE and analyzed by immunoblotting. The Phos-tag ligand (20 µM) and two equivalents of MnCl2 (40 µM) were added to the separating gel before polymerization. The following primary antibodies were used: rabbit anti-histone H3 phospho-Ser10 (1:1,000; #3377, CST, Danvers, MA, USA), rabbit anti-histone H3 phospho-Thr3 (1:100,000; ab78351, Abcam, Cambridge, UK), rabbit anti-SMC2 (1:500),10 mouse anti-INCENP (1:1,000),28 rabbit anti-TopoIIα (1:500),29 and mouse anti-beta-actin (1:20,000; GT5512, GeneTex, Irvine, CA, USA). The secondary antibodies were as follows: IRDye 800CW donkey anti-rabbit IgG (1:10,000; 926-32211, Li-Cor Biosciences, 6 ACS Paragon Plus Environment
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Lincoln, NE, USA), or anti-mouse IgG (1:10,000; 926-32210, Li-Cor Biosciences). Immune complexes were detected using an Odyssey CLx Infrared Imaging System and the ImageStudio 5.2 software (Li-Cor Biosciences). Trypsin digestion and phosphopeptide enrichment Proteins associated with mitotic chromosomes were extracted by PTS,26 and 100-µg aliquots were digested using 1 µg endoproteinase Lys-C (Wako) and 0.4 µg trypsin (Promega, Fitchburg, WI, USA) in 2 mM DTT and 10 mM IAA for 16 h. Digestion was stopped with 1% TFA followed by desalting using the SDB-XC stage tip.30
Digested
phosphopeptides
were
enriched
by
TiO2-based
hydroxy
acid-modified metal oxide chromatography (HAMMOC) followed by desalting with the SDB-XC tip.31 Mass spectrometry The enriched phosphopeptides were analyzed by LC-MS using a Q Exactive mass spectrometer (Thermo Fisher Scientific) coupled to HPLC using a 25 cm C18 column via a nanoelectrospray ion source. The linear gradient ranged from 5% to 40% buffer B (buffer A = 5% acetonitrile in 0.1M trifluoroacetic acid, buffer B = 80% acetonitrile in 0.1M trifluoroacetic acid) in 240 min. MS2 scan was obtained for the 10 most intense peaks of each MS1 scan. The individual biological replicas were measured multiple times (4 times in Exp1, 3 in Exp2, 2 in Exp3, and 1 in Exp4). Peptide identification and quantification For identification of phosphorylation sites, peak lists were created using Andromeda
32
and ProteoWizard
33
based on the recorded fragmentation spectra.
Peptides and proteins were identified by automated database search using MASCOT search engine (Matrix Science, UK) against Gallus gallus protein database in UniProt (release 2013_07) and our in-house chicken database, with a precursor mass tolerance of 10 ppm, a fragment ion mass tolerance of 0.02 Da, and strict trypsin specificity (cleavage next to Lys or Arg but not Arg-Pro) allowing for up 7 ACS Paragon Plus Environment
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to 2 missed cleavages. Peptides were considered identified if the Mascot score was over the 95% confidence limit (p
