BRCA2 Phosphorylated by PLK1 Moves to the Midbody to Regulate Cytokinesis Mediated by Nonmuscle Myosin IIC

Published OnlineFirst January 21, 2014; DOI: 10.1158/0008-5472.CAN-13-0504

Cancer Tumor and Stem Cell Biology Research

Miho Takaoka1, Hiroko Saito2, Katsuya Takenaka1, Yoshio Miki1,2, and Akira Nakanishi1

Abstract Cytokinesis is the critical ﬁnal step in cell division. BRCA2 disruption during cytokinesis is associated with chromosome instability, but mechanistic information is lacking that could be used to prevent cancer cell division. In this study, we report that BRCA2 phosphorylation by the mitotic polo-like kinase (PLK1) governs the localization of BRCA2 to the Flemming body at the central midbody, permitting an interaction with nonmuscle myosin IIC (NM-IIC). Formation of an NM-IIC ring-like structure at the Flemming body shows that the IIC-ring relies on its ATPase activity stimulated by interaction with BRCA2 and associated proteins. Notably, inhibiting this binding inactivated the ATPase activity, causing disassembly of the IIC-ring, defective formation of the midbody, and interruption of cytokinesis. An analysis of cancer-associated mutations in BRCA2 at the PLK1-binding site suggests that they may contribute to cytokinetic defects by altering BRCA2 localization. Our ﬁndings suggest that BRCA2-dependent IIC-ring formation is a critical step in proper formation of the midbody, offering an explanation for how chromosome instability may arise in breast cancer. Cancer Res; 74(5); 1–11. 2014 AACR.

Introduction polo-box domain (PBD), which binds to phosphopeptides Germline mutations in the BRCA2 gene have been reported within a consensus motif of S-[pS/pT]-[P/X] (15, 16). The PBD to increase the risk of developing breast and ovarian cancer. regulates cellular function, the interaction with substrates, and The BRCA2 protein has multiple functions, including DNA the subcellular localization of Plk1 (17). Plk1 is also required for double-strand break repair (1–3), and the regulation of cen- appropriate localization of substrates (18). Previous studies trosome amplification and localization (4, 5). BRCA2 also revealed that Plk1 binds to the N-terminal region of BRCA2 and contributes to the regulation of cytokinesis (6, 7). During phosphorylates Ser193, and that this phosphorylation is fi anaphase, constriction of the actomyosin ring leads to forma- enhanced as mitosis progresses (19, 20). However, the speci c tion of a cleavage furrow (8–10). Continued furrowing results in Plk1-binding site within this region of BRCA2 has not been fi the formation of a narrow intercellular bridge, which contains identi ed. The role of BRCA2 phosphorylation in the mainte- the midbody, consisting of a bundled microtubule and a ring- nance of genome stability also remains unclear. like structure called the Flemming body within its central Nonmuscle myosin-II (NM-II) proteins in humans are hex- portion (11). Several studies have implicated various protein amers, consisting of a pair of heavy chains and two pairs of light factors in the modulation of cytokinesis by BRCA2 (12–14). chains that hydrolyze MgATP. They are members of a family of Disruption of BRCA2 during cytokinesis leads to disorganiza- actin-binding motor proteins that play essential roles in cel- tion of myosin-II at the cleavage furrow and the intercellular lular processes such as cell division and embryonic develop- bridge (6). ment. The NM-II family comprises three isoforms: NM-IIA, Mitotic polo-like kinase 1 (Plk1) is a key regulator of mitosis NM-IIB, and NM-IIC. These contain different nonmuscle myo- from mitotic initiation to cytokinesis. Plk1 contains a serine/ sin heavy chains (NMHC-IIA, NMHC-IIB, and NMHC-IIC) that MYH 9, MYH10 threonine kinase domain followed by the carboxy-terminal are encoded by (myosin heavy chain) , and MYH14, respectively (21, 22). The N-terminal region of the NMHC-II protein consists of a globular head containing the Authors' Affiliations: 1Department of Molecular Genetics, Medical actin-binding region, an ATPase domain, and a Src homology 3 Research Institute, Tokyo Medical & Dental University, Bunkyo-Ku; and (SH3)-like domain (myosin head; refs. 23, 24). NM-IIC is 2Department of Genetic Diagnosis, the Cancer Institute of JFCR, Koto-Ku, Tokyo, Japan alternatively spliced both in loop-1 and loop-2. Isoform NM- IIC0 contains no inserts in either of the loops. An 8–amino acid Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). extension in the loop-1 region is present in isoforms NM-IIC1 and NM-IIC1C2. Isoform NM-IIC1C2 displays a 33–amino acid Corresponding Author: Yoshio Miki, Department of Molecular Genetics, Medical Research Institute, Tokyo Medical & Dental University, Yushima 1- extension in the loop-2 region. The presence of 8 amino acid 5-45, Bunkyo-Ku, Tokyo 113-8510, Japan. Phone: 81-3-5803-5825; Fax: insert in NM-IIC increases the actin-activated ATPase activity 81-3-5803-0242; E-mail: [email protected] (25). The C-terminal deletion isoform of NM-IIC is already doi: 10.1158/0008-5472.CAN-13-0504 present in the Ensembl database (http://www.ensembl.org/ 2014 American Association for Cancer Research. index.html; MYH14-007, Protein ID: ENSP00000469573). Jana

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and colleagues reported the localization of NM-IIC to the Results midbody during the process of abscission. In that report, they Localization of BRCA2 to the Flemming body through confirmed that NM-IIC1 is required for cytokinesis (25). phosphorylation by Plk1 Accordingly, we made use of this isoform. In this study, we To analyze the role of phosphorylation of BRCA2 Ser193 by suggest that BRCA2-dependent IIC-ring formation represents Plk1, we generated an antibody (193pBRCA2) specific for this a key step in proper midbody formation, and that a hereditary phosphoprotein and confirmed the specificity of the breast cancer-associated mutation within the Plk1 interaction 193pBRCA2 antibody by performing a competition assay using motif of BRCA2 affects the localization of BRCA2 to the pS193-Peptides and treatment with protein phosphatase (PP2A; Flemming body, resulting in cytokinesis defects. Supplementary Fig. S1A and S1B). This antibody recognized Ser193-phosphorylated BRCA2 (pS193-BRCA2) in anti-BRCA2 Materials and Methods (Ab-1) immunoprecipitates from midbody lysates (Supple- Detailed descriptions of plasmids, transfections, siRNA mentary Fig. S1C).We observed its localization during the cell fl treatment, antibodies, immunofluorescence and three-dimen- cycle by immuno uorescence microscopy. BRCA2 localized to sional (3D) reconstitutions, immunoprecipitation, Western the midbody (particularly at the Flemming body) during fi blot analysis, binding assays, glycerol density gradient centri- cytokinesis of mitotic HeLaS3 cells (Fig. 1A). Similar ndings fugation, time-lapse microscopy, measurement of ATPase were observed in A549, MCF7, and U2OS cells (Supplementary activity, cell-cycle analysis, and statistical analysis are provided Fig. S1D). The phosphorylated BRCA2 also localized to the in the Supplementary Materials and Methods. Flemming body (Fig. 1A). In contrast, BRCA1 did not localize to the Flemming body (Fig. 1A and Supplementary Fig. S1E). This Cell culture result is consistent with that of a previous study by another HeLaS3 and COS-7 cells were purchased from the RIKEN group (13). We then isolated the midbodies from HeLaS3 cells GENBANK, and U2OS and A549 were from American Type (Supplementary Fig. S1F) and compared the extent of BRCA2 Culture Collection (ATCC). These cells were cultured in Dul- phosphorylation in midbody lysates with that in whole-cell becco's Modified Eagle Medium containing 10% fetal calf lysates (Fig. 1B). A greater fraction of the BRCA2 was phos- fl serum (FCS). MCF7 was purchased from ATCC and cultured phorylated in the midbody. Using immuno uorescence 193 in Eagle's Minimum Essential Medium containing 10% FCS, microscopy, we showed that Plk1 colocalizes with pS - 0.01 mg/mL bovine insulin, and 1% sodium pyruvate. The BRCA2 at the Flemming body (Fig. 1C). Assays of COS-7 cells identities of HeLaS3, U2OS, A549, and MCF7 cell lines were transfected with BRCA2-FLAG and Plk1-HA demonstrated confirmed by short tandem repeat (STR) profiling by BEX CO., that BRCA2 and Plk1 coimmunoprecipitated (Fig. 1D). We – Ltd and used within 6 months of testing. COS-7 cells (monkey located a Plk1-binding motif (S-T-P sequence at codons 76 78) cell line) were not analyzed by STR profiling because STR in the N-terminal region of BRCA2. A Thr77-phosphorylated – profiling is method for authentication of human cell lines. peptide corresponding to this region (a.a. 73 82 of BRCA2) was synthesized for in vitro binding analyses. Streptavidin bead– Midbody isolation immobilized p-Thr77 peptide precipitated Plk1 from mitotic Mitotic cells (A549) were released from colcemid treatment HeLaS3 cell lysates, whereas unphosphorylated peptide or (50 ng/mL) for 17 hours by washing twice with fresh medium. peptide-free streptavidin beads did not (Fig. 1E). Plk1 was not After incubating at 37C for 60 minutes, the cells were collect- precipitated from S-phase lysates due to low expression. ed. Midbody isolation was performed according to the meth- This Plk1-binding site is conserved among diverse species ods of Mullins and McIntosh (26). (Supplementary Fig. S1G). A hereditary breast cancer-associated missense mutation (T77A) within this site is annotated in Measurement of ATPase activity the Breast Cancer Information Core (BIC) database (http:// COS-7 cells were transfected with plasmids expressing research.nhgri.nih.gov/bic/). We constructed plasmids encod- HA-tagged NMHC-IIC and FLAG-tagged BRCA2. After 24 ing a truncated N-terminal region [BRCA2 (R1)-FLAG (WT):1– hours, cells (5 106) were lysed in lysis buffer [20 mmol/L 157 a.a.] containing either the wild-type Plk1-binding site or Tris-HCl (pH 8.0), 100 mmol/L NaCl, 1 mmol/L EDTA, 1% this mutant, BRCA2 (R1)-FLAG (T77A; Supplementary Fig. NP-40, and protease inhibitors]. BRCA2 and NMHC-IIC S1H). The mutant protein was unable to interact with endog- were immunoprecipitated by anti-FLAG or anti-HA anti- enous Plk1 in HeLaS3 cells (Supplementary Fig. S1I), indicating bodies, respectively. Each sample was eluted by 40 mL that this mutation disrupted the interaction with Plk1. Next, we elution buffer (50 mmol/L glycine, pH 2.8), which was examined whether overexpression of this truncated region immediately neutralized with 1 mol/L Tris. BRCA2-FLAG could compete with endogenous BRCA2. Coimmunoprecipita- and NMHC-IIC-HA were mixed and incubated at room tion assays were performed in HeLaS3 cells expressing BRCA2 temperature for 1 minute and then placed into a solution (R1)-FLAG (WT). It was demonstrated that this region inhibited for measurement of ATPase activity. F-actin was added the binding of endogenous BRCA2 to Plk1 (Supplementary Fig. immediately before the measurement. ADP production S1J). Also, the endogenous BRCA2 ceased to be detected at the during ATP hydrolysis was measured as a change in NADH Flemming body following ectopic expression of this region concentration. NADH absorbance was measured every 2 (Supplementary Fig. S1K). These results suggested a domi- seconds at 340 nm and the rate of NADH consumption was nant-negative effect of HA-BRCA2 (R1) upon endogenous interpreted as the ATPase activity. BRCA2. A distinct HA-tagged BRCA2 region [HA-BRCA2 (R2):

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BRCA2 Regulates Cytokinesis by Myosin-IIC ATPase Activation

A Interphase Prophase MetaphaseAnaphase Cytokinesis

193pBRCA2

BRCA2

BRCA1

B C 193pBRCA2 Plk1 Merge

(IB) Total whole- cell lysate Midbody lysate Ser193- 380 phosphorylated 193pBRCA2 BRCA2 BRCA2 380 BRCA2 (Ab-1) (with Hoechst) kDa D E streptavidin S-phase Input (IP) FLAG ** QLASpTPIIEK Bead + or Plk1-HA Mitotic HeLa S3 lysate biotin BRCA2-FLAG QLASpTPIIEK QLASTPIIEK HA +++ FLAG (IB) 75 HA Plk1-HA S-phase S-phase S-phase Mitosis Mitosis S-phase Mitosis Mitosis FLAG BRCA2 75 kDa 380 -FLAG Plk1 kDa 50 kDa

Figure 1. Plk1-phosphorylated BRCA2 localizes to the Flemming body in HeLaS3 cells. A, cells were ﬁxed and stained for Ser193-phosphorylated BRCA2 (pS193-BRCA2), BRCA2, and BRCA1 throughout the cell cycle. Nuclei were detected by Hoechst 33258 stain (blue). Arrows, the Flemming body. Scale bar, 5 mm. B, total whole-cell and midbody lysates were subjected to Western blot analysis using antibodies against pS193-BRCA2 and BRCA2. IB, immunoblot. C, cells were ﬁxed and stained for pS193-BRCA2 (green) and Plk1 (red) in cytokinesis. White arrows, the Flemming body. Scale bar, 5 mm. D, COS-7 cells were transfected with Plk1-HA and/or BRCA2-FLAG expression plasmids. The expression of transgene-derived proteins in input cell lysates and anti-FLAG immunoprecipitates (IP) analyzed using anti-HA or anti-FLAG antibodies are shown. E, N-terminal biotin-fused pThr77 peptides (QLASpTPIIEK) were linked to streptavidin beads and tested for the ability to precipitate Plk1 from S-phase or mitotic HeLaS3 cell lysates. The phosphorylated Thr77 residue and the Ser76 residue crucial for PBD binding are indicated with asterisks (). Immunoblot analyses show Plk1 coprecipitated with the peptides.

112–685 a.a.] did not affect the localization of the endogenous localization to the Flemming body and that a cancer-associated BRCA2 (Supplementary Fig. S1K). To evaluate further the effect mutation of BRCA2 might prevent phosphorylation leading to of BRCA2 phosphorylation at Ser193 upon its localization, we its mislocalization. expressed either wild-type recombinant BRCA2-FLAG or mutants thereof containing substitutions of Ser193 by alanine Interaction between BRCA2 and NM-IIC (S193A) or glutamate (S193E). Although both the wild-type and BRCA2 deﬁciency causes abnormal accumulation of myo- the S193E mutant localized to the Flemming body, the S193A sin-II, particularly during cytokinesis (6). However, the phys- mutant did not (Supplementary Fig. S1L). This suggested that iologic role of colocalization of BRCA2 and myosin-II to the phosphorylation of BRCA2 at Ser193 by Plk1 is important for its midbody remains unclear. We ﬁrst examined the subcellular

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localization of each NM-II isoform in A549 cells during cyto- ﬁbers, that is, exhibited a nonbundled midbody and lacked kinesis (Fig. 2A). NM-IIC localized to the Flemming body and the Flemming body (Fig. 3B, bottom). The fraction of abnormal formed a ring-like structure. In contrast, NM-IIA was diffu- cells (exhibiting a non-Flemming body) was 25.2 3.6% in the sively distributed throughout the midbody, and NM-IIB was control cells and 63.5 3.6% in the knockdown cells (Fig. 3B). localized within an area from the basal portion of the midbody At 48 hours, we observed a 1.5- to 3-fold increase in the number to the cytosol of the dividing cell. Similar results were obtained of multinuclear cells in response to NMHC-IIC knockdown in human mammary epithelial cells (Supplementary Fig. S2A). (IIC-1: 57.2 4.5%; IIC-2: 36.3 1.7%) compared with control Further analysis of 3D images by confocal laser microscopy cells (scrambled control: 21.0 2.0%; no-transfection: 15.8 clearly demonstrated that NM-IIC formed part of a unique 4.5%; Supplementary Fig. S3A). The NMHC-IIC knockdown ring-like structure within the central portion of the midbody cells were observed using time-lapse differential interference (Supplementary Fig. S2B). This structure, which we term the microscopy for 48 hours after siRNA treatment. A subpopula- IIC-ring, was approximately 1.5 mm in diameter and coloca- tion of the cells had fused back together, resulting in binucle- lized with the Flemming body. To characterize this BRCA2- ated cells. Another subpopulation exhibited catastrophic cell NM-IIC colocalization further, we analyzed its sedimentation death before cytokinesis completion (Supplementary Fig. S3B). in a glycerol gradient following isolation from A549 cells (Fig. This process is shown in greater detail by time-lapse images 2B and Supplementary Fig. S2C). The pS193-BRCA2, NM-IIC, (Supplementary Fig. S3C). Plk1, and MgcRacGAP (a marker of the midbody) were all detected within fraction 2, whereas NM-IIB was detected in Inhibition of IIC-ring formation by BRCA2 gene silencing fraction 3, and NM-IIA was present in several fractions (1–8). To see an effect of BRCA2 suppression on the IIC-ring BRCA1 was not found at the midbody and nonphosphorylated formation, we silenced BRCA2 using siRNA in A549 cells, BRCA2 was mainly detected within fraction 4 (Supplementary which express all three isoforms: IIA, IIB, and IIC (Fig. 3C). Fig. S2C). These results suggested that pS193-BRCA2 might As observed in the NMHC-IIC knockdown cells, midbody interact with NM-IIC and MgcRacGAP in addition to Plk1 at ﬁbers were present in the absence of the Flemming body in the Flemming body. BRCA2-FLAG and NMHC-IIC-HA were the BRCA2 knockdown cells. Consequently, NM-IIC was coexpressed in COS-7 cells and cell lysates were subjected to distributed throughout the nonbundled midbody, and the immunoprecipitation with anti-FLAG and anti-HA antibodies. IIC-ring was not apparent (Fig. 3D and Supplementary Fig. NMHC-IIC-HA and BRCA2-FLAG were both detected in the S3D).Agreaternumberofabnormal midbodies were anti-FLAG or anti-HA immunoprecipitates (Fig. 2C). Endoge- observed in siRNA-BRCA2–treated cells (siRNA-BRCA2-1: nous BRCA2 immunoprecipitates from A549 cells were shown 48.3 2.9%; siRNA-BRCA2-3: 43.3 7.6%) than in control to contain NMHC-IIC (Fig. 2D and Supplementary Fig. S2D). cells (siRNA-luciferase: 21.7 2.9%; no-transfection: 10.0 This result was validated by two different antibodies that 5.0%; P ¼ 0.017; Fig. 3D, right). Similar results were recognized distinct epitopes of NMHC-IIC (Fig. 2D) and by obtained in U2OS cells treated with siRNA targeting BRCA2 mass spectrometric analysis (Supplementary Fig. S2D). Immu- (Supplementary Fig. S3E and S3F). To determine whether noprecipitations from A549 cell lysates using anti-BRCA2 BRCA2 is important for the formation of midbody struc- antibody revealed the presence of several protein components tures and progression of cytokinesis, we analyzed the in the BRCA2 complex. Polypeptides with apparent molecular BRCA2 knockdown HeLa S3 cells undergoing cytokinesis. weights of 485, 385, 220, 156, and 122 kDa coimmunoprecipi- BRCA2 protein levels were reduced after siRNA application tated with BRCA2 (Supplementary Fig. S2D). As a control, we (Fig. 3E), and the length of the midbody during cytokinesis also performed immunoprecipitation with normal rabbit in HeLa S3 cells was greater in BRCA2 siRNA-treated cells immunoglobulin G (IgG). These polypeptides were digested than in control cells (siRNA-luciferase or no-transfection; P in gel by trypsin, and the resulting peptides were sequenced by < 0.01; Fig. 3F). Next, we examined whether the length of nanoelectrospray mass spectrometry. Besides NMHC-IIC and the midbody in siRNA-BRCA2 cells could be restored by BRCA2, polycystin-1 (PKD1), ATM, Rho-associated protein expressing BRCA2-FLAG or BRCA2 (S193A)-FLAG (Supple- kinase 1 (ROCK1), and PARP1 were coimmunoprecipitated by mentary Fig. S3G and S3H). Although BRCA2-FLAG local- the anti-BRCA2 antibody (Supplementary Fig. S2D). In con- ization was observed in the midbody, this was not seen in trast, NM-IIB did not bind to BRCA2 in this assay. Endogenous BRCA2 (S193A)-FLAG cells (Supplementary Fig. S3H, left). MgcRacGAP did not also bind to BRCA2 or NM-IIC (Supple- When BRCA2-FLAG was expressed, the length of the mid- mentary Fig. S2E and S2F) despite its colocalization to the body was similar to that in the control (siRNA-luciferase). Flemming body. In contrast, the length of the midbody did not recover in cells expressing BRCA2 (S193A)-FLAG (P ¼ 0.041; Supple- Abnormal cytokinesis following NMHC-IIC gene mentary Fig. S3H, right), suggesting that it could not silencing localize to the midbody. We did not observe an abnormal To investigate the role of the IIC-ring during cell division, we midbody or IIC-ring in cells treated with the BRCA1-siRNA observed the formation of the midbody 24 hours following (Supplementary Fig. S4A–S4E). These results suggest that siRNA knockdown of NMHC-IIC (Fig. 3A). Midbody structures the formation of the midbody and IIC-ring may require the in most siRNA-control cells were bundled and formed the presence of BRCA2, but not BRCA1, at the Flemming body Flemming bodies (Fig. 3B, top). In contrast, a majority of the and that the binding of BRCA2 to NM-IIC is necessary for cells treated with the NMHC-IIC siRNA retained midbody this process.

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BRCA2 Regulates Cytokinesis by Myosin-IIC ATPase Activation

A NMHC-IIC with midbody marker NMHC-IIC NMHC-IIC

NMHC-IIA

Plk1 MgcRacGAP

NMHC-IIB

Merge Merge

NMHC-IIC

Flemming body NMHC α-tubulin Hoechst

B C Midbody lysate 35% Glycerol:15% (IP) Input 1 2 3 4 5 6 7 8 9 10 11 12 13 14 NMHC-IIC-HA 380 pS193 BRCA2 BRCA2-FLAG 250 HA NMHC-IIC FLAG (IB) 250 FLAG 380 NMHC-IIA IP; HA 250 250 HA NMHC-IIB kDa IP; FLAG

75 MgcRacGAP

75 Plk1

TOPO2α 150 kDa

D (IP) (IP) (IP) (IP) Input Input Input Input BRCA2 BRCA2 Mouse IgG Mouse IgG BRCA2 Mouse IgG BRCA2 Mouse IgG

250 250 250 380 kDa kDa kDa kDa (IB) NMHC-IIC-N NMHC-IIC-C NMHC-IIB BRCA2

Figure 2. NM-IIC localizes to the Flemming body and interacts with BRCA2. A, immunoﬂuorescence in A549 cells stained for NMHC-IIA (green), IIB (green), IIC (green), and a-tubulin (red). Cells in cytokinesis were also costained for NMHC-IIC (green or red) and Plk1 (red) or MgcRacGAP (green). Scale bar, 5 mm. B, midbody lysates from A549 cells were separated by 15% to 35% (w/v) glycerol gradient centrifugation and subjected to Western blot analysis using the indicated antibodies. The nuclear marker, TOPO2-a, was not detected in any fractions. C, COS-7 cells were transfected with the indicated expression plasmids. The expression of transgene-derived proteins in cell lysates, anti-FLAG immunoprecipitates (IP), and anti-HA immunoprecipitates (IP) revealed by anti-FLAG or anti-HA antibodies are shown. D, A549 cell lysates were immunoprecipitated (IP) with either mouse IgG or anti-BRCA2 antibodies and immunoprecipitates were analyzed using polyclonal antibodies to different epitopes of NMHC-IIC at the N-(NMHC-IIC-N) and C-(NMHC-IIC-C) terminals. Samples were also analyzed by anti-BRCA2 and anti–NMHC-IIB antibodies.

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ABsiRNA (10 nmol/L) siRNA-control 80 P = 0.0002 IIC-1 IIC-2 SC NT (IB) Flemming body 250 NMHC-IIC 60 250

NMHC-IIA 40 siRNA-IIC 250 20 NMHC-IIB

50 0 β-Actin Non-Flemming body cell number (%) IIC SC 37 siRNA kDa

CDα-Tubulin NMHC-IIC Merge A549 1 P = 0.017 60 siRNA- 12 BRCA2 2 40 3 34 siRNA- 20 4 luciferase siRNA-luciferase siRNA-luciferase NT siRNA-BRCA2-1 siRNA-BRCA2-1 siRNA-BRCA2-3 Abnormal midbody (%) 5 0 380 BRCA2 5 6 NT 50 NT 6 BRCA2-1 β-actin BRCA2-3Luciferase 37 With Hoechst siRNA kDa

siRNA-BRCA2-1 EF 30 P < 0.01 HeLa S3 P < 0.01 siRNA μ m) 20 Midbody (μm)

siRNA-luciferase BRCA2-1 BRCA2-2 BRCA2-3 Luciferase NT 10 380 BRCA2

50 β-Actin ( distance Midbody 37 0 kDa α -Tubulin BRCA2-1 BRCA2-3 Luciferase NT siRNA

Figure 3. NM-IIC and BRCA2 are essential for midbody formation in A549 cells. A, lysates from A549 cells treated with NMHC-IIC siRNA (IIC-1 or IIC-2) or siRNA-scramble (SC), or no-transfection (NT) were subjected to Western blot analysis using anti–NMHC-IIC, IIA, IIB, and anti–b-actin antibodies. B, NMHC-IIC siRNA (bottom) and siRNA-scramble (top)-treated cells were observed by phase contrast microscopy. The bar graph shows the mean fractions ( SD) of cells showing abnormal midbodies based on three independent experiments (n ¼ 100). C, lysates from A549 cells treated with two different BRCA2 siRNAs or siRNA-luciferase or no-transfection (NT) were subjected to Western blot analysis with anti-BRCA2 and anti–b-actin antibodies. D, A549 cells treated with BRCA2 siRNA or siRNA-luciferase or siRNA-no-transfection were fixed and stained with anti–a-tubulin (red) and anti–NMHC-IIC antibodies (green) in cytokinesis. Scale bar, 5 mm. Magnifications of dotted square areas are shown on the right (1–6). The bar graph shows the mean frequencies (SD) of abnormal midbodies in cells based on three independent experiments (n ¼ 20). E, lysates from HeLa S3 cells treated with three different BRCA2 siRNAs or siRNA- luciferase or no-transfection (NT) were subjected to Western blot analysis with anti-BRCA2 and anti–b-actin antibodies. F, HeLa S3 cells treated with BRCA2 siRNA (BRCA2-1) were fixed and stained for a-tubulin (red). White lines, the measured midbody length by Adobe Photoshop CS3 software. Scale bar, 5 mm. The midbody lengths are shown in box plot that signifies the top and bottom quartiles. The medians are represented by bold lines. Nonparametric data were statistically analyzed by the Mann–Whitney U test (n ¼ 81 in each case, P < 0.01).

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A NMHC-IIC Myosin head (motor domain) Myosin tail Figure 4. The binding inhibition 1 2003 a.a. Binding between BRCA2 and NM-IIC strength ATP-binding site affects the formation of Flemming NMHC-IIC ( 1–2003 a.a. ) + body. A, a schematic diagram of IIC (A) ( 1–1000 a.a. ) +++ NMHC-IIC deletion mutants [IIC IIC (A1) ( 1–341 a.a. ) ++ (A), (B), (A1), (A2), and (A3)] and the IIC (A2) (331–1670 a.a. ) + strength of binding to BRCA2, IIC (A3) (661–1000 a.a. ) + summarizing the data within IIC (B) (887–2003 a.a. ) – Supplementary Fig. S5A and S5B. (–; none, +; weak, ++; intermediate, +++; strong) NMHC-IIC fragments were fused to an HA tag. B, COS-7 cells B (IP) C (IP) BRCA transfected with indicated Input FLAG plasmids were lysed and proteins BRCA2-FLAG were immunoprecipitated with NMHC-IIC-HA (IB) HA anti-FLAG antibody. Inputs and IIC(A1)-HA IIC(A1)-HA kDa precipitates (IP) were visualized by 250 HA kDa NMHC-IIC anti-HA and anti-FLAG antibodies. (IB) 250 C, A549 cells were transfected with HA BRCA2 380 either IIC (A1)-HA or HA and 50 FLAG 380 HA midbodies were collected. Anti- 37 BRCA2 immunoprecipitates (IP) from midbody lysates were D NMHC-IIC HA BRCA2 HA analyzed by anti–NMHC-IIC, anti- BRCA2, and anti-HA antibodies. D, IIC (A1) A549 cells were transfected with -HA IIC (A1)-HA or HA and stained with anti–NMHC-IIC (green) or BRCA2 (green), and anti-HA (red) antibodies. Scale bar, 5 mm. HA

Binding of BRCA2 to the N-terminal region of NMHC-IIC sing control cells (Supplementary Fig. S5D; P < 0.01 by Mann– To determine which region of NMHC-IIC interacts with Whitney U test). The IIC (A1)-HA–expressing cells were unable BRCA2, we introduced plasmids encoding HA-tagged to divide and accumulated at G1 phase (76.09%–48.89% for the NMHC-IIC deletion mutants (Fig. 4A) and BRCA2-FLAG into cells transfected with the empty vector; Supplementary Fig. COS-7 cells, and performed coimmunoprecipitation analyses. S5E). These results indicated that the binding of BRCA2 to NM- BRCA2 bound to the IIC (A)-HA fusion, containing a.a. 1–1,000 IIC could have a function in cytokinesis promotion. of NMHC-IIC (Supplementary Fig. S5A). We further divided Because the endogenous BRCA2 did not localize to the this fragment into three partially overlapping regions and Flemming body in A549 cells expressing the R1 region of found that only the IIC (A1)-HA fusion bound strongly to BRCA2 (Supplementary Fig. S1K), we supposed that the abun- BRCA2-FLAG (Supplementary Fig. S5B). When IIC (A1)-HA dant expression of the R1 domain would also inhibit IIC-ring was coexpressed with BRCA2-FLAG and NMHC-IIC-HA, the formation. As expected, the IIC ring was not observed in A549 interaction between BRCA2-FLAG and NMHC-IIC-HA was cells expressing a high level of HA-BRCA2 (R1), as was the case abolished (Fig. 4B). The same dominant-negative effect was in cells expressing IIC (A1)-HA (Fig. 4D and Supplementary Fig. seen between the endogenous BRCA2 and NM-IIC at the S5C and S5F). A greater number of binucleated cells was midbody (Fig. 4C). Both the localization of endogenous BRCA2 observed in BRCA2 (R1)-FLAG–expressing cells (18.5 and IIC-ring formation at the Flemming body were disrupted in 3.4%) than in FLAG-expressing control cells (8.3 2.7%; A549 cells expressing IIC (A1)-HA (Fig. 4D and Supplementary Supplementary Fig. S5G). Fig. S5C). Enhancement of the ATPase activity of NM-IIC by BRCA2 NM-II plays a fundamental role in cell adhesion, migration, Abnormalities in cytokinesis induced by expression of and division mediated by inherent actin cross-linking and NMHC-IIC (A1) or BRCA2 (R1) contractile functions. This activity requires energy, which is We hypothesized that the interaction between BRCA2 and provided by the hydrolysis of ATP and the active site of NM-II NM-IIC at the midbody might play a role in the regulation of exhibits ATPase activity. Treatment of A549 cells with bleb- cytokinesis. To explore this possibility, we analyzed A549 cells bistatin, an inhibitor of myosin II ATPase activity, caused decay exposed to anti–a-tubulin antibody 48 hours after IIC (A1)-HA of the IIC-ring surrounding a-tubulin and imperfections in ring transfection. The midbodies of the IIC (A1)-HA–expressing formation (Supplementary Fig. S6A). MgcRacGAP was cells varied in length compared with those of the HA-expres- observed in the decay of the IIC-ring (Supplementary Fig.

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1 2 3 4 5 6 7 8 9 10 A 2.5

) 2.0 –3 1.5

1.0 ATPase activity (unit/min: ×10 0.5

0 0.63 0.63 0.95 0.95 0.95 0 0 0 0 0.63 NMHC-IIC-HA (μg) 000000000.630 NMHC-IIC (AA)-HA (μg) 0.50 0.75 0.75 0.75 0.75 0.50 0.75 0.75 0.75 0 BRCA2-FLAG (μg) 0 0 0 1.60 0 0 0 1.60 0 0 IIC (A1)-HA (μg) ++++–+++ + + F-actin (1.3 μg)

siRNA- B C siRNA-BRCA2 NMHC-IIC-GFP control NT 1; ATP + M g2+ 2; ATP + M g2+(blebbistatin) a d e c

b f NMHC-IIC-GFP 3; ATP 4; M g2+ D BRCA2-FLAG/ FLAG/ BRCA2-FLAG/ NMHC-IIC-GFP NMHC-IIC-GFP GFP

GFP

abcdefBRCA2

Merge

Figure 5. ATPase activity is required for IIC-ring formation. A, the actin-dependent ATPase activity of NM-IIC following incubation of the immunoprecipitated NMHC-IIC-HA with and without BRCA2-FLAG. B, NMHC-IIC-GFP was expressed in COS-7 cells and the reaction mixtures were mounted on a glass slide and observed by fluorescence microscopy. The IIC-rings were not seen following blebbistatin treatment or lacking either ATP or Mg2þ in the reaction. Magnified images of NMHC-IIC-GFP are also shown (a–f). C, NMHC-IIC-GFP was expressed in COS-7 cells treated with siRNA-BRCA2 or siRNA-control or no- transfection, and the reaction mixtures were mounted on a glass slide and observed by fluorescence microscopy. The samples were fixed and stained for GFP (green). Scale bar, 1 mm. D, the plasmids indicated in figure were expressed in COS-7 cells and the cell lysates were mounted on a glass slide. The samples were fixed and stained for GFP (green) and BRCA2 (red). Scale bar, 1 mm.

S6B). To explore the function of BRCA2 in IIC-ring formation, were present (0.5 mg BRCA2-FLAG and 0.63 mg NMHC-IIC- we analyzed the effect of BRCA2 on the actin-dependent HA; Fig. 5A, lane 1). ATPase activation following incubation of ATPase activity of NM-IIC. The actin-dependent ATPase activ- NMHC-IIC and BRCA2 occurred in a dose-dependent manner ity of NM-IIC was measured following incubation of the (Fig. 5A, lanes 1 and 2). The light chain was phosphorylated immunoprecipitated NMHC-IIC-HA in the presence or following the addition of BRCA2-FLAG to the immunopreci- absence of BRCA2-FLAG. We conﬁrmed that introduction of pitated NMHC-IIC (Supplementary Fig. S6D). Furthermore, in the plasmid encoding NMHC-IIC into cells leads to binding of the presence of IIC (A1)-HA, which inhibits the binding of the exogenous NMHC-IIC to the 12A and 12B isoforms of BRCA2 to NM-IIC, ATPase activation was inhibited (Fig. 5A, endogenous light chain (Supplementary Fig. S6C). The ATPase lane 4). The absence of F-actin from the reaction mixture was activated (1.0 10 3 unit/minute) when both proteins reduced ATPase activity signiﬁcantly (Fig. 5A, lane 5). The

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BRCA2 Regulates Cytokinesis by Myosin-IIC ATPase Activation

Phosphorylated BRCA2 induced by Plk1

Figure 6. The midbody abscission is Midbody modulated by the BRCA2–NM-IIC NMNM IICIIC ATPaseAT activityty NM IIC:with complex. The phosphorylation of fforor IIIC-ringIC-r formationon elevated AATPaseTPase activityact BRCA2 by Plk1 is required for Dissociation AAssociationssociation BRCA2 localization to the NM IIC:with low Flemming body and may support NM IICIC BRCA2BRCA2 NM IIC BRCA2 ATPase activity IIC-ring formation through ( A1 overover expression)expression) PhPhosphorylatedosphoryl increased NM-IIC-ATPase activity. BRBRCA2CA2 IfIf ATPaseATPass activity When BRCA2-NM-IIC binding NMHC IIC (A1) inhibition induced by IIC (A1) ofof NNMM IIICIC is inhibited…d… overexpression, the activation “Fail in activation” BlebbistatinBlebbis “Activation”“Activation” of NM-IIC ATPase failed and the MutantMutant of ATP IIC-ring was not formed normally as bbindinginding site it would be with treatment with blebbistatin (ATPase inhibitor of myosin) to the cells. The IIC-ring is triggered by BRCA2-dependent binding of NM-IIC at the Flemming body, which may allow BRCA2 to Tubulinn modulate the midbody abscission No IIC-ring formation IIIC-ringIC-ring for normal transition of cells → BRCA2 siRNA IIC-ring formation through cytokinesis. → abscission failure NMHC IIC siRNA abscission success

localization of F-actin at the Flemming body was observed by ization of BRCA2 in the lysates. We showed that BRCA2 immunofluorescence microscopy (Supplementary Fig. S6E). A localized to the IIC ring-like structure (Fig. 5D). These results mutant NMHC-IIC (AA)-HA in which Lys204 and Thr205 in the suggest that the ring-like structure is composed of NMHC-IIC- ATP-binding site (GESGAGKT; 198–205) were substituted with GFP and BRCA2. alanine did not exhibit ATPase activity in response to incubation with BRCA2 (Fig. 5A, lane 9). IIC (A1)-HA also did not The requirement of the ATPase activity of NM-IIC for exhibit ATPase activity (Fig. 5A, lane 8). A mutant BRCA2 IIC-ring formation (S193A)-FLAG also supported activation of NM-IIC ATPase To demonstrate the significance of the ATPase activity of NM- activity to a level similar to that observed by the wild-type IIC for IIC ring formation, we examined whether the IIC-ring BRCA2-FLAG (Supplementary Fig. S6F). These results sug- could be restored by recombinant NMHC-IIC-HA or NMHC-IIC gested that the ATPase activity is increased by NM-IIC–BRCA2 (AA)-HA expression following endogenous NMHC-IIC knock- association. Furthermore, the phosphorylation of BRCA2- down (Supplementary Fig. S6G and S6H). Both were copreci- Ser193 is not essential for the activation of the ATPase activity pitated with BRCA2-FLAG (Supplementary Fig. S6I). Although of NM-IIC but is required for the localization of BRCA2 to the cells expressing wild-type NMHC-IIC-HA exhibited IIC-ring Flemming body. within the midbody, in cells expressing NMHC-IIC (AA)-HA, To test this further, we attempted an in vitro reconstitution the IIC-ring-like structure seemed to degenerate within the of the IIC-ring using recombinant NMHC-IIC. NMHC-IIC-GFP midbody area (Supplementary Fig. S6J). These results indicate was expressed in COS-7 cells and examined using the cell that the ATPase activity of NM-IIC is required for IIC-ring lysates. It was observed that NMHC-IIC-GFP formed part of a formation. unique ring-like structure (1.4–2.0 mm) in the presence of both þ ATP and Mg2 (Fig. 5B, 1). However, NMHC-IIC-GFP failed to organize into a ring-like structure when blebbistatin was added Discussion to the reaction (Fig. 5B, 2). The ring-like structure was also not BRCA2, the product of breast cancer susceptibility gene þ observed in the absence of either ATP or Mg2 (Fig. 5B, 3 and 4). BRCA2, plays important roles in the maintenance of genome To see an effect of BRCA2 on IIC-ring formation, we over- stability throughout the cell cycle. Several studies have sug- expressed NMHC-IIC-GFP following siRNA-mediated knock- gested a role for BRCA2 in regulation of cytokinesis at the late down of BRCA2 in COS-7 cells. NMHC-IIC-GFP failed to M phase. BRCA2 deficiency impairs completion of cell division. organize into a ring-like structure in siRNA-BRCA2–treated Inhibition of cell separation is accompanied by abnormalities cells (Fig. 5C). Next, we coexpressed recombinant NMHC-IIC- in NM-II organization during the late stages of cytokinesis (6). GFP and BRCA2-FLAG in COS-7 cells and investigated local- We identified the subcellular localization of each isoform

www.aacrjournals.org Cancer Res; 74(5) March 1, 2014 OF9

Takaoka et al.

during cytokinesis and found that only the NM-IIC isoform light chain, resulting in the activation of ATPase activity. colocalizes and interacts with BRCA2 at the Flemming body ROCK, which phosphorylates Ser19 of smooth muscle MLC2, (Fig. 2 and Supplementary Fig. S2). The interaction of NM-IIC was detected in the anti-BRCA2 immunoprecipitates by mass with BRCA2 at the Flemming body allows the activation of NM- spectrometry. This result raises the possibility that ROCK1 may IIC ATPase activity followed by IIC-ring formation (Fig. 6). phosphorylate and activate myosin IIC. This issue should be Cytokinesis, the final step of cell division in all animal cells, addressed in future studies. partitions the cytoplasm between two daughter cells. This BRCA2 is a multifunctional protein that functions as a process depends upon the activity of NM-II, which participates caretaker of genome stability throughout the cell cycle, includ- in the formation of the cleavage furrow. Attachment of a ing during processes such as DNA synthesis, centrosome contractile ring, consisting of a network of actin filaments dynamics, chromosome separation, and cytokinesis. In this (actomyosin) and NM-II, to the cytoplasmic membrane study, we demonstrated a role for BRCA2 in the late stage of induces the formation of a cleavage furrow. Mammalian cells cytokinesis in collaboration with NM-IIC, which should help use not IIC but IIA or IIB during this process (10). NM-IIA clarify the mechanics of cytokinesis and breast oncogenesis. exhibited a diffuse distribution throughout the entire midbody We identified in the BIC database a missense mutation in as observed by immunofluorescence microscopy (Fig. 2A) and BRCA2 (T77A, within the Plk1-binding motif), the clinical glycerol gradient analyses (Fig. 2B), indicating that it might significance of which had not been established. We showed interact with various proteins. Although NM-IIB localizes to an the possibility that this mutation may abolish the binding area from the basal portion of the midbody to the cytosol of between BRCA2 and Plk1, resulting in failure of BRCA2 local- dividing cells, glycerol gradient analysis revealed that it is ization to the Flemming body and cytokinesis incompletion, absent from the fraction containing phosphorylated BRCA2. suggesting that this mutation may underlie the development of In addition, a coimmunoprecipitation assay did not detect breast cancer. interaction between BRCA2 and NM-IIB (Fig. 2D). In contrast, fl NM-IIC was detected at the Flemming body and it colocalized Disclosure of Potential Con icts of Interest No potential conflicts of interest were disclosed. with phosphorylated BRCA2 during cytokinesis just before abscission. Depletion of BRCA2 or NM-IIC by siRNA disrupted Authors' Contributions the IIC-ring and led to abnormal midbody formation followed Conception and design: Y. Miki, A. Nakanishi by failure of abscission and cytokinesis (Fig. 3 and Supplemen- Development of methodology: M. Takaoka, Y. Miki, A. Nakanishi Acquisition of data (provided animals, acquired and managed patients, tary Fig. S3). Cells expressing NM-IIC (A1), which exhibited a provided facilities, etc.): M. Takaoka, A. Nakanishi dominant-negative effect upon the interaction of endogenous Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): M. Takaoka, A. Nakanishi BRCA2 and NM-IIC, also failed to progress through cytokinesis Writing, review, and/or revision of the manuscript: M. Takaoka, K. Take- (Fig. 4 and Supplementary Fig. S5C–S5E). Taken together, the naka, Y. Miki, A. Nakanishi interaction between BRCA2 and NM-IIC seems essential for Administrative, technical, or material support (i.e., reporting or orga- nizing data, constructing databases): M. Takaoka, H. Saito, Y. Miki, A. abscission or the induction thereof. Some cells, including HeLa Nakanishi S3, have been reported not to express NM-IIC; however, we Study supervision: K. Takenaka, c, A. Nakanishi have performed some experiments to confirm NM-IIC-expression in HeLa S3 cells. These include Western blot data using Acknowledgments – The authors thank Rie Hayashi (Leica Microsystems K.K.) for support in anti NMHC-IIC-N antibody and the mass spectrometric anal- preparing the laser-scanning confocal microscope. ysis of HeLa S3-cell lysates (data not shown). These data indicated the existence of the short type of NM-IIC in HeLa Grant Support S3 cells. This work was supported by JSPS KAKENHI Grant Numbers 24300328 In this study, the interaction of BRCA2 with NM-IIC was (Y. Miki), 25640061 (Y. Miki), 21790311 (K. Takenaka), 23590355 (K. Takenaka), by Ishidsu Shun Memorial Scholarship (M. Takaoka), and by Takeda Science shown to be required for the activation of NM-IIC ATPase Foundation (K. Takenaka). activity (Fig. 5A and Supplementary Fig. S6F). Furthermore, the The costs of publication of this article were defrayed in part by the payment of endogenous light chain bound to NMHC-IIC-HA was phos- page charges. This article must therefore be hereby marked advertisement in phorylated following the addition of BRCA2-FLAG (Supple- accordance with 18 U.S.C. Section 1734 solely to indicate this fact. mentary Fig. S6C and S6D). We speculate that a kinase present Received February 18, 2013; revised December 20, 2013; accepted January 5, in the BRCA2 immunoprecipitate might phosphorylate the 2014; published OnlineFirst January 21, 2014.

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BRCA2 Phosphorylated by PLK1 Moves to the Midbody to Regulate Cytokinesis Mediated by Nonmuscle Myosin IIC

Miho Takaoka, Hiroko Saito, Katsuya Takenaka, et al.

Cancer Res Published OnlineFirst January 21, 2014.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-13-0504

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