Cancer Research CTRC-AACR San Antonio Breast Cancer Symposium
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Futamura, M.
Right arrow Articles by Nakamura, Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Futamura, M.
Right arrow Articles by Nakamura, Y.
[Cancer Research 60, 1531-1535, March 15, 2000]
© 2000 American Association for Cancer Research

Advances in Brief

Potential Role of BRCA2 in a Mitotic Checkpoint after Phosphorylation by hBUBR11

Manabu Futamura, Hirofumi Arakawa, Koichi Matsuda, Toyomasa Katagiri, Shigetoyo Saji, Yoshio Miki and Yusuke Nakamura2

Laboratory of Molecular Medicine, Human Genome Center, Institute of Medical Science, University of Tokyo, Tokyo 108-8639 [M. F., H. A., K. M., T. K., Y. N.]; Second Department of Surgery, Gifu University School of Medicine, Gifu 500-8705 [M. F., S. S.]; Department of Molecular Diagnosis, Japanese Foundation for Cancer Research, Tokyo 170 [Y. M.], Japan


   ABSTRACT
Top
 ABSTRACT
Introduction
 Materials and Methods
 Results and Discussion
 REFERENCES
 
BRCA2, a gene responsible for inherited susceptibility to breast cancer in a number of families, is thought to be critical for replication and repair of DNA during S-phase. To elucidate the physiological functions of BRCA2, we used a yeast two-hybrid system to screen for proteins that could associate with BRCA2. Here we report interaction of BRCA2 with a mitotic checkpoint protein, hBUBR1, and its phosphorylation by hBUBR1 in vitro. After cotransfection of BRCA2 and hBUBR1 expression vectors into the COS7 cell line, both proteins were stained together in the nuclei of cells whose spindle fibers were disrupted, but not in undamaged cells. Treatment with vincristine, which disrupts microtubules, significantly increased expression of both hBUBR1 and BRCA2 in the MCF7 cells. The results suggest that BRCA2 protein might be involved in a mitotic checkpoint in vivo after it has been phosphorylated by hBUBR1.


   Introduction
Top
 ABSTRACT
 Introduction
Materials and Methods
 Results and Discussion
 REFERENCES
 
Approximately 5–10% of breast cancers can be attributed to inheritance of genetic defects that have occurred in certain genes (1) . Among the genes conferring susceptibility to breast cancer, BRCA1 and BRCA2 already have been isolated (2 , 3) , and germline mutations of one or the other have been identified in ~80% of patients with familial breast cancer (4 , 5) . To date, frameshifts that yield truncated products, presumably nonfunctional, have been the most common mutations identified (6 , 7) . Tumors from individuals carrying germline BRCA1 or BRCA2 mutations often have lost the wild-type allele, suggesting that the absence of functional BRCA1 or BRCA2 proteins is a direct cause of early-onset breast cancer (8) .

The BRCA2 gene encodes a large molecule (3418 amino acids) that bears no significant sequence homology to any other known protein (3 , 9) . In mammalian cells, BRCA2 interacts with Rad51, a homologue of Escherichia coli RecA, which plays key roles in homologous recombination and DNA repair after double-strand breakage (10, 11, 12) . Moreover, deleted versions of the murine Brca2 gene cause increased sensitivity to DNA damage and spontaneous accumulation of chromosomal abnormalities in mice, but they do not impair checkpoint function or apoptosis in response to DNA damage (12) . Despite these clues, however, the cellular function of BRCA2 has remained uncertain.

In yeast, chromosomal stability and segregation are monitored and maintained by a set of proteins that includes Bub1, Bub2, Bub3, Mad1, Mad2, Mad3, and Mps1 (13) . Damage to mitotic spindle fibers can activate these mitotic checkpoint genes, whose products arrest mitosis and repair the spindles (13 , 14) . Mutations in any of these genes result in failure to arrest the cell cycle at G2-M, and cells exit mitosis prematurely (14) . Inactivation of murine BUB1 also impairs cell-cycle arrest in the mouse, causing premature cell-cycle progression and aneuploidy (15) . These facts suggest that mitotic checkpoint genes may be critical for preventing aneuploidy during chromosomal segregation of mammalian cells.

To investigate the function of BRCA2, we have used a yeast two-hybrid system to search for proteins in addition to Rad51 that can interact with BRCA2. Here we report the detection of one such protein, hBUBR1 (16) . We also demonstrate that this association can lead to phosphorylation of BRCA2 protein.


   Materials and Methods
Top
 ABSTRACT
 Introduction
 Materials and Methods
Results and Discussion
 REFERENCES
 
Yeast Two-Hybrid Screening.
We applied a yeast two-hybrid screening system (17) to isolate cDNAs that encode proteins that interact with the R7 domain (amino acids 2861–3176) of BRCA2. We constructed a vector by subcloning BRCA2-R7 into the EcoRI-XhoI site of pAS2–1 (Clontech). This vector was used as "bait" to screen an oligo(dT)-primed human testis cDNA library in pACT2 vector (Clontech), according to the manufacturer’s instructions. Positive clones were cotransfected into yeast with either the bait vector or the original pAS2–1 vector; ß-galactosidase activity served as a marker to confirm the interaction.

Sequence Analysis and Homology Search.
Each clone obtained by yeast two-hybrid screening was sequenced by the dideoxy nucleotide termination method using an ABI 377 DNA sequencer (Perkin-Elmer). These sequences were tested against known genes in the public database with the BLAST and FASTA programs (18 , 19) .

Construction of Vector Expressing hBUBR1.
The entire coding sequence of hBUBR1 was subcloned into the pcDNA3.1 (Invitrogen) expression vector. To construct myc- or HA-tagged hBUBR1, an oligonucleotide duplex that encodes Kozac and epitope sequences was inserted into the HindIII-EcoRI site of pcDNA3.1. hBUBR1 cDNAs were ligated in-frame into the EcoRI-XhoI site of myc- or HA-pcDNA. Both constructs were confirmed by sequencing. For in vitro translation, BRCA2-R7 with an encoded Kozac sequence was also inserted into the HindIII-EcoRI site of pcDNA3.1.

Cell Culture and Transfection.
COS7 cells cultured in DMEM containing 10% fetal bovine serum and antibiotic/antimycotic solution (Sigma) were plated in 10-cm culture dishes (1 x 106 cells/dish) 24 h before transfection. Expression vectors of hBUBR1 (4 µg/dish) were transfected using LIPOFECTAMINE PLUS (Life Technologies, Inc.), according to the manufacturer’s instructions. Cells were harvested 24 h after transfection.

Immunoprecipitation.
Transfected COS7 cells were collected and lysed in a lysis buffer [150 mM NaCl, 50 mM Tris-HCl (pH 8.0), 1% NP40, 1 µg/ml phenylmethylsulfonyl fluoride]. Lysates were precleared with protein G-Sepharose, and then incubated with anti-myc (Oncogene Research) or anti-HA (Boehringer Mannheim) antibody at 4°C for 2 h. Immune complexes were precipitated with 15 µl of protein G-Sepharose and washed five times with lysis buffer.

Expression of Fusion Proteins in E. coli.
Three cDNA fragments containing the COOH terminus of the coding sequence of hBUBR1 (Fig. 1Citation ; GST3 -hBUBR1A, amino acids 786-1050; GST-hBUBR1B, amino acids 676-1050; GST-hBUBR1C, full size, amino acids 1–1050) and BRCA2-R7 were amplified by PCR using Pfu DNA polymerase (Stratagene). The products were ligated in-frame into pGEX5X-1 (for hBUBR1) or pGEX5X-2 (for BRCA2; Pharmacia), and confirmed by sequencing. Overnight cultures of XL1 Blue MRF transformed with plasmids encoding GST-fusion proteins were diluted 10-fold with fresh medium and incubated for 3–4 h at 37°C. After recombinant proteins were induced with 0.1 mM isopropyl-1-thio-ß-D-galactopyranoside for 3–4 h at 30°C, harvested cells were lysed by sonication in PBS containing 1% Triton X-100. The lysates were clarified by centrifugation, and then rotated with Glutathione-Sepharose 4B (Pharmacia) for 2 h at 4°C. The beads were washed five times with sonication buffer, and the proteins were eluted in buffer containing 50 mM Tris-HCl (pH 8.0) and 10 mM glutathione.



View larger version (8K):
[in this window]
[in a new window]
  		Fig. 1. Constructs of GST-fused hBUBR1 proteins. hBUBR1 (1050 amino acids; top) has two conserved domains, CD1 and CD2, that are highly homologous to S. cerevisiae BUB1. CD2 includes a kinase domain. The three horizontal bars indicate the regions of the hBUBR1 protein that were used for making bacterial expression vectors GST-hBUBR1A, GST-hBUBR1B, and GST-hBUBR1C. GST-hBUBR1A (265 amino acids) is the same region of the clone initially obtained from yeast two-hybrid screening, which lacks the NH2-terminal part of the kinase domain. GST-hBUBR1B (375 amino acids) and GST-hBUBR1C (1050 amino acids) express the kinase domain and the full-size protein of hBUBR1, respectively.

 
In Vitro Protein-binding Assay.
After equilibration with lysis buffer, the GST-hBUBR1A fusion protein immobilized to Glutathione-Sepharose 4B was mixed with in vitro-translated, 35S-labeled BRCA2 protein synthesized using the TNT T7-coupled reticulocyte lysate system (Promega). The mixture was incubated overnight at 4°C with rotation, and then washed six times with the lysis buffer described above. The precipitates were boiled in SDS sample buffer for 3 min, and eluted proteins were analyzed by SDS-PAGE on a 12% gel. Autoradiography was performed for 3 h at room temperature.

In Vitro Kination Assay.
Immunoprecipitates and immobilized GST-fusion proteins (GST-hBUBR1B and C) were equilibrated in kinase buffer [50 mM NaCl, 50 mM HEPES (pH 7.4), 20 mM ß-glycerophosphate, 10 mM MgCl2, 10 mM MnCl2, 1 mM sodium vanadate, 1 mM DTT, and 1 mM phenylmethylsulfonyl fluoride], and then incubated with 10 µCi of 32P-{gamma}ATP (Amersham) and substrate at 30°C for 30 min (20) . Histone H1 protein (Life Technologies) was used as a positive control. Each sample was boiled in SDS sample buffer for 3 min, and eluted proteins were analyzed by SDS-PAGE on an 8% gel. Autoradiography was performed for several hours at -80°C with intensifying screens.

Microinjection and Immunocytochemistry.
A BRCA2 expression vector (pCDNA3_HA/BRCA2 CORRECTtr2) was kindly provided by Dr. David M. Livingston (Dana-Farber Cancer Institute, Boston, MA). COS7 cells (1 x 106) were plated in 10-cm culture dishes; 24 h later, myc-tagged hBUBR1 and BRCA2 expression vectors (0.5 µg/ml of each) were injected into at least 103 COS7 cells by microinjection (Eppendorf). These cells were treated with vincristine 24 h after injection, fixed 24 h later with PBS containing 4% paraformaldehyde, and then rendered permeable with PBS containing 0.1% Triton X-100 for 2.5 min at 4°C. Cells were incubated with blocking solution (3% BSA and 0.25% goat serum in PBS) for 1 h at room temperature, and then anti-myc mouse monoclonal antibody (PharMingen) diluted 1:1000 in blocking solution and/or anti-BRCA2 goat polyclonal antibody (1:1000 dilution in blocking solution; Santa Cruz Biotechnology Inc.) for 1 h at room temperature. Antibodies were stained with a horse antimouse secondary antibody conjugated to Texas Red and/or a rabbit antigoat secondary antibody conjugated to FITC, and viewed with an ECLIPSE E800 microscope (Nikon).

Isolation of RNA and Northern Blot Analysis.
Total RNAs were prepared from the cell lines treated with vincristine using TRIzol (Life Technologies). Poly(A)+ RNA was purified from total RNA with Oligotex-dT30 (JSR, Japan). A 2-µg aliquot of each poly(A)+ RNA was separated on a 1% agarose gel containing 1x 4-morpholinosulfonic acid and 2% formaldehyde and transferred to nylon membrane. The blots were hybridized with a random-primed 32P-labeled hBUBR1, BRCA2, and ß-actin cDNA probe in a mixture of 5x saline-sodium phosphate-EDTA and 10x Denhardt’s-2% SDS-50% Formamide at 50°C, washed with 0.1x SSC-0.1% SDS at 65°C, and exposed for autoradiography at -80°C.


   Results and Discussion
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results and Discussion
REFERENCES
 
During a search by two-hybrid screening for proteins capable of associating with BRCA2, we isolated several candidate clones that revealed specific interaction with the R7 domain of BRCA2 (amino acids 2861–3176) and determined their sequences. One 1193-bp cDNA included an open reading frame encoding a 265-amino acid peptide. Database analysis using BLAST and FASTA programs indicated that the predicted product was identical to the COOH-terminal sequence of hBUBR1 protein (16) .

Recently published data have implied that BRCA2 may be involved in a mitotic checkpoint that maintains accurate chromosomal segregation and guards against chromosomal instability. For example, a targeted mutation to truncate mBrca2 at exon 11 resulted in an accumulation of chromosomal abnormalities in the mouse, including breaks and aberrant chromatid exchanges (12) . However, the precise mechanism by which inactivation of this gene causes chromosomal instability is not clear. On the other hand hBUBR1, a human homologue of Saccharomyces cerevisiae BUB1, appears to monitor accurate chromosomal segregation and cell division during mitosis (16) . In yeast, mutations of BUB1 lead to abnormal numbers of chromosomes, suggesting an important role of this protein in maintenance of genomic stability (14) . hBUBR1 contains a kinase domain at the COOH terminus, and phosphorylates itself (20) . Defects in cell-cycle checkpoints may be responsible for the genomic instability of cancer cells (13 , 21) . On the basis of that information, we considered that by associating with hBUBR1, BRCA2 might prevent abnormal chromosomal segregation. Thus, we decided to confirm its ability to interact with hBUBR1, a known mitotic checkpoint protein.

We performed a pull-down assay to examine whether the BRCA2-R7 and hBUBR1A proteins would interact in vitro. Using a nucleotide sequence from the clone initially isolated by yeast two-hybrid screening, we synthesized the GST-fusion protein (GST-hBUBR1A; 56 kDa) in bacteria and immobilized it on Glutathione-Sepharose 4B (Fig. 2A)Citation . LacZ (119 kDa) and BRCA2-R7 (34 kDa) proteins, translated in vitro and labeled with 35S (Fig. 2B)Citation , were incubated with GST or purified GST-hBUBR1A proteins. After intensive washing, the pelleted beads were dissolved in SDS-loading buffer and analyzed by SDS-PAGE electrophoresis. Neither LacZ nor BRCA2-R7 had coprecipitated with the GST protein itself (Fig. 2CCitation , Lanes 1 and 2), but the 35S-labeled BRCA2-R7 peptide clearly had coprecipitated with GST-fused hBUBR1A (Fig. 2CCitation , Lane 4).



View larger version (50K):
[in this window]
[in a new window]
  		Fig. 2. Interaction of the R7 domain of BRCA2 with GST-hBUBR1A in vitro. A, Coomassie Brilliant Blue staining of GST and GST-hBUBR1A proteins used for the in vitro binding assay. An arrow indicates GST-hBUBR1A protein, which showed a molecular mass of 56 kDa. B, in vitro-translated, 35S-labeled LacZ (119 kDa) and BRCA2-R7 (34 kDa) proteins. C, in vitro binding assay. GST (Lanes 1 and 2) and GST-hBUBR1A (Lanes 3 and 4) proteins were incubated with either in vitro-translated LacZ (Lanes 1 and 3) or BRCA2-R7 (Lanes 2 and 4). After intensive washing, pelleted proteins were subjected to 12% SDS-PAGE followed by Coomassie Brilliant Blue staining and autoradiography. A and C were obtained from the same gel.

 
The region of hBUBR1 that interacts with BRCA2-R7 is considered to be its kinase domain. Moreover, the yeast homologue, BUB1, is known to exert a checkpoint function by phosphorylating target proteins (13) . Hence, we speculated that hBUBR1 might phosphorylate BRCA2, and performed an in vitro kination assay to investigate that possibility. GST-hBUBR1B and GST-hBUBR1C proteins (see Fig. 1Citation ) were incubated with GST-BRCA2-R7 protein or GST-protein in the presence of [{gamma}-32P]ATP. A 61-kDa band representing phosphorylated GST-BRCA2-R7 (Fig. 3ACitation , arrows) was detected, in addition to bands of 67 and 139 kDa that corresponded to autophosphorylated GST-hBUBR1B and GST-hBUBR1C, respectively (Fig. 3ACitation , *). Incubation of GST-BRCA2-R7 protein with GST-protein in this assay did not cause phosphorylation of GST-BRCA2-R7 (data not shown).



View larger version (53K):
[in this window]
[in a new window]
  		Fig. 3. In vitro kination assay. A, phosphorylation of BRCA2-R7 peptide by GST-hBUB1B and GST-hBUB1C. GST-hBUB1B and GST-hBUB1C proteins were incubated with either GST-protein (27 kDa; Lane 1) or GST-BRCA2-R7 protein (61 kDa; Lane 2). After incubation, aliquots of the mixtures were subjected to 12% SDS-PAGE followed by Coomassie Brilliant Blue staining and autoradiography. The images of Coomassie Brilliant Blue staining (left gel in each panel) and autoradiography (right gel in each panel) were obtained from the same gel. Autophosphorylated versions of both GST-hBUB1B (67 kDa) and GST-hBUB1C proteins (139 kDa) were seen in all lanes (*). Arrows indicate phosphorylated GST-fused BRCA2-R7 proteins; the phosphorylated band of ~75 kDa is nonspecific. B, phosphorylation of GST-BRCA2-R7 in COS7 cells by hBUBR1 immune complexes precipitated by anti-myc or anti-HA antibodies. hBUBR1 protein complexes purified from lysates of COS7 cells transfected with myc- or HA-tagged hBUBR1 expression vectors were incubated with GST (27 kDa; Lane 1), GST-BRCA2-R7 protein (61 kDa; Lane 2), no protein (Lane 3), or histone H1 (33 kDa; Lane 4) in the presence of [{gamma}-32P]ATP. After incubation, aliquots of each mixture were subjected to 12% SDS-PAGE followed by Coomassie Brilliant Blue staining and autoradiography. The images of Coomassie Brilliant Blue staining (left gel in each panel) and autoradiography (right gel in each panel) were obtained from the same gel. Autophosphorylation of myc- or HA-tagged hBUBR1 proteins (112 kDa) was observed in all lanes (*). Arrows indicate phosphorylated GST-BRCA2-R7 protein.

 
We also investigated the phosphorylation of BRCA2-R7 by hBUBR1 protein in mammalian cells. myc- or HA-tagged hBUBR1 expression vectors were transfected into COS7 cells. hBUBR1 protein was precipitated from the cell extracts by anti-myc or anti-HA antibodies, and incubated with GST-BRCA2-R7 protein as a substrate. In addition to 139-kDa phosphorylated bands, we observed 61-kDa (Fig. 3BCitation , Lane 2, arrow) and 35-kDa (Fig. 3BCitation , Lane 4) bands that corresponded to phosphorylated GST-BRCA2-R7 and histone H1 (positive control). Interestingly, the intensity of the bands decreased when hBUBR1 protein was incubated with GST-BRCA2-R7 (Fig. 3BCitation , Lane 2) or histone H1. These results implied that the immune complex that included hBUBR1 precipitated by HA- or myc-antibody could actually phosphorylate GST-BRCA2-R7 protein. On the basis of these results, we suggest that BRCA2 is likely to be a substrate of hBUBR1 in vivo. Incubation of GST-BRCA2-R7 protein with the precipitates that formed with myc- or HA-monoclonal antibody from the mock-transfected cell lysate did not influence phosphorylation status of GST-BRCA2-R7 (data not shown).

BRCA2 is now well known as an important player in the S-phase, where it is involved in replication and recombination processes associated with DNA repair (10, 11, 12) . On the other hand, hBUBR1 is thought to play a significant role in a spindle-damage checkpoint during M-phase (22 , 23) . These observations seem inconsistent with our results that indicate interaction between these two proteins. However, because hBUBR1 does phosphorylate the BRCA2-R7 domain in vitro and because disruption of microtubules activates hBUBR1 kinase activity, we considered that hBUBR1 might phosphorylate BRCA2 in cells that had sustained damage to microtubules. Therefore, we examined the locations of hBUBR1 and BRCA2 within cells treated with vincristine, a drug that disrupts the microtubule structure. In this series of experiments, COS7 cells transfected with hBUBR1 and/or BRCA2 expression vectors were treated with vincristine for 24 h, fixed, and stained with anti-BRCA2 (Fig. 4A)Citation or anti-myc (Fig. 4B)Citation antibodies. When the cells were damaged with vincristine to arrest the cell-cycle in M-phase, BRCA2 was costained in the nuclei with hBUBR1 (Fig. 4CCitation , arrows). The nuclear staining pattern of BRCA2 in damaged cells was diffuse and very different from that in undamaged cells. In the cells that probably were not much affected by vincristine and therefore not arrested at M-phase (Fig. 4CCitation , without arrows), BRCA2 appeared as a spotty green stain in the nucleus (24) . We estimated that 40% of the cells were damaged and that 60% were undamaged under the condition of Fig. 4Citation on the basis of the morphology of the cells and the fluorescence-activated cell-sorting data. Almost all damaged cells revealed nuclear costaining of BRCA2 and hBUBR1 similar to that shown in Fig. 4CCitation . This result suggests that damage to microtubules activates mitotic checkpoint genes, including BUBR1, causing a drastic change in the cellular location of BRCA2 protein as it becomes phosphorylated.



View larger version (34K):
[in this window]
[in a new window]
  		Fig. 4. Nuclear staining of hBUBR1 and BRCA2 in cells arrested in mitosis by a microtubule-disrupting drug. myc-tagged hBUBR1 expression vector and BRCA2 expression vector were microinjected into COS7 cells; 24 h later, the cells were treated with vincristine. After an additional 24 h of incubation, the drug-treated cells were fixed and stained with anti-myc and anti-BRCA2 antibodies. A, staining with goat anti-BRCA2 polyclonal antibody. Spotty and diffuse types of nuclear staining of BRCA2 protein are shown, respectively, in interphase and mitotic-arrested cells. B, hBUBR1 protein stained with anti-myc mouse monoclonal antibody. Perinuclear and diffuse cytoplasmic staining of hBUBR1 protein is shown in interphase cells, whereas strong nuclear staining of hBUBR1 was induced by vincristine in cells arrested in mitosis (round shape). C, visual overlay of COS7 cells double-stained with anti-myc mouse monoclonal antibody and anti-BRCA2 goat polyclonal antibody. Arrows indicate mitotic cells arrested by vincristine. Both proteins were stained diffusely in nuclei of these cells. D, DAPI staining of nuclei.

 
We examined the inducibility of BRCA2 by serial dosage (0.0001–1 µM) of vincristine and determined that BUBR1 was induced by vincristine in a dose-dependent manner and that BRCA2 was also induced by a dosage of 0.001–1 µM of vincristine (Fig. 5)Citation . Similar results were obtained in experiments using 293 cell lines (data not shown). To exclude the possibility that BRCA2 protein might accumulate in the nuclei of normal mitotic cells, we examined the expression and cellular localization of BRCA2 in M-phase by Northern analysis and by immunostaining in combination with a standard cell-cycle synchronization technique. We detected no significant increase of BRCA2 mRNA, but we did see the spotty staining pattern characteristic of BRCA2 protein in normal mitotic cells (data not shown). These results clearly indicate that BRCA2 might play a previously undocumented role in cells with damaged microtubules, and might be involved in a mitotic checkpoint along with hBUBR1.



View larger version (44K):
[in this window]
[in a new window]
  		Fig. 5. Induction of BRCA2 and hBUBR1 mRNA by microtubule damage. MCF7 cells were treated with serial dosages of vincristine; 24 h later, RNAs were extracted from harvested cells. Levels of BRCA2 and hBUBR1 mRNA expression were assessed by Northern blot. ß-actin was run as a control.

 
We have demonstrated here that hBUBR1 interacts with and phosphorylates the BRCA2-R7 domain in vitro, and that a microtubule-disrupting drug can induce mRNA expression of both of BRCA2 and hBUBR1; this process causes simultaneous translocation of hBUBR1 from the cytoplasm to the nucleus and relocation of BRCA2 in the nucleus from spotty to diffuse. To date, a number of studies that focused on the role of BRCA2 in the S-phase (25 , 26) have determined only that BRCA2 might play an important role in DNA-damage repair with the participation of RAD51 (10) . However, no evidence has explained a mechanism to account for the spontaneous accumulation of chromosomal abnormalities in cells lacking wild-type Brca2, including breaks and aberrant chromatid exchanges. Our findings provide significant clues for clarifying this phenomenon, leading us to speculate that hBUBR1 monitors accurate chromosomal segregation through phosphorylation of target proteins, of which BRCA2 protein is probably one. Although the actual outcome of BRCA2 phosphorylation is still uncertain, mutational loss of the function of BRCA2 might block hBUBR1 signaling pathways and result in genomic instability as a consequence of failure of a mitotic checkpoint.


   ACKNOWLEDGMENTS
 
We thank Dr. David M. Livingston for providing the BRCA2 expression vector.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported in part by a Grant-in-Aid from the Ministry of Education, Science, Sports and Culture of Japan, and by "Research for the Future" Program Grant 96L00102 of the Japan Society for the Promotion of Science. Back

2 To whom requests for reprints should be addressed, at Human Genome Center, Institute of Medical Science, University of Tokyo, Shirokanedai, Minato-ku, Tokyo 108-8639, Japan. Phone: 81-3-5449-5372; Fax: 81-3-5449-5433; E-mail: yusuke{at}ims.u-tokyo.ac.jp Back

3 The abbreviation used is: GST, glutathione S-transferase. Back

Received 10/13/99. Revised 12/30/99. Accepted 1/28/00.


   REFERENCES
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results and Discussion
 REFERENCES
 

  1. Stratton M. R., Wooster R. Hereditary predisposition to breast cancer. Curr. Opin. Genet. Dev., 6: 93-97, 1996.[Medline]
  2. Miki Y., Swensen J., Shattuck-Eidens D., Futreal P. A., Harshman K., Tavtigian S., Liu Q., Cochran C., Bennett L. M., Ding W., Bell R., Rosenthal J., Hussey C., Tran T., McClue M., Frye C., Hattier T., Phelps C., Haugen-Strano A., Katcher H., Yakumo K., Gholami Z., Shaffer D., Stone S., Bayer S., Wray C., Bogden R., Dayananth P., Ward J., Tonin P., Narod S., Bristow P. K., Norris F. H., Helvering L., Morrison P., Rosteck P., Lai M., Barrett J. C., Lewis C., Neuhausen S., Cannon-Albright L., Goldgar D., Wiseman R., Kamb A., Skolnick M. H. A strong candidate for the breast and ovarian cancer susceptibility gene BRCA1. Science (Washington DC), 266: 66-71, 1994.[Abstract/Free Full Text]
  3. Wooster R., Bignell G., Lancaster J., Swift S., Seal S., Mangion J., Collins N., Gregory S., Gumbs C., Micklem G., Barfoot R., Hamoudi R., Patel S., Rice C., Biggs P., Hashim Y., Smith A., Conner F., Arason A., Gudmundsson J., Ficenec D., Kelsell D., Ford D., Tonin P., Bishop T., Spurr N. K., Ponder B. A. J., Eeles R., Peto J., Devilee P., Cornelisse C., Lynch H., Narod S., Lenoir G., Egilsson V., Barkadottir R. B., Easton D. F., Bantley D. R., Futreal P. A., Ashworth A., Stratton M. R. Identification of the breast cancer susceptibility gene BRCA2. Nature (Lond.), 378: 789-792, 1995.[Medline]
  4. Breast Cancer Linkage Consortium. Pathology of familial breast cancer: differences between breast cancers in carriers of BRCA1 or BRCA2 mutations and sporadic cases. Lancet, 349: 1505-1510, 1997.[Medline]
  5. Ford D., Easton D. F., Stratton M., Narod S., Goldgar D., Devilee P., Bishop D. T., Webr B., Lenoir G., Chang-Claude J., Sobol I. I., Teare M. D., Struewing J., Arason A., Schereck S., Peto J., Rebbeck T. R., Tonin P., Neuhausen S., Barkardottir R., Eyfjord J., Lynch H., Ponder B. A., Gayther S. A., Zelada-Hedman M. Genetic heterogeneity and penetrance analysis of the BRCA1 and BRCA2 genes in breast cancer families. The Breast Cancer Linkage Consortium. Am. J. Hum. Genet., 62: 676-689, 1998.[Medline]
  6. Roa B. B., Boyd A. A., Volcik K., Richards C. S. Ashkenazi Jewish population frequencies for common mutations in BRCA1 and BRCA2. Nat. Genet., 14: 185-187, 1996.[Medline]
  7. Fodor F. H., Weston A., Bleiweiss I. J., McCurdy L. D., Walsh M. M., Tartter P. I., Brower S. T., Eng C. M. Frequency and cancer risk associated with common BRCA1 and BRCA2 mutations in Ashkenazi Jewish breast cancer patients. Am. J. Hum. Genet., 63: 45-51, 1998.[Medline]
  8. Abeliovich D., Kaduri L., Lerer I., Weinberg N., Amir G., Sagi M., Zlotogora J., Heching N., Peretz T. The founder mutations 185delAG and 5382insC in BRCA1 and 6174delT in BRCA2 appear in 60% of ovarian cancer and 30% of early-onset breast cancer patients among Ashkenazi women. Am. J. Hum. Genet., 60: 505-514, 1997.[Medline]
  9. Tavtigian S. V., Simard J., Rommens J., Couch F., Shattuck-Eidens D., Neuhausen S., Merajver S., Thorlacius S., Offit K., Stoppa-Lyonnet D., Belanger C., Bell R., Berry S., Bogden R., Chen Q., Davis T., Dumont M., Frye C., Hattier T., Jammulapati S., Janecki T., Jiang P., Kehrer R., Leblanc J. F., Goldgar D. E. The complete BRCA2 gene and mutations in chromosome 13q-linked kindreds. Nat. Genet., 12: 333-337, 1996.[Medline]
  10. Sharan S. K., Morimatsu M., Albrecht U., Lim D. S., Regel E., Dinh C., Sands A., Eichele G., Hasty P., Bradley A. Embryonic lethality and radiation hypersensitivity mediated by Rad51 in mice lacking Brca2. Nature (Lond.), 24: 804-810, 1997.
  11. Mizuta R., LaSalle J. M., Cheng H. L., Shinohara A., Ogawa H., Copeland N., Jenkins N. A., Lalande M., Alt F. W. RAB22 and RAB163/mouse BRCA2: proteins that specifically interact with the RAD51 protein. Proc. Natl. Acad. Sci. USA, 94: 6927-6932, 1997.[Abstract/Free Full Text]
  12. Patel K. J., Vu V. P., Lee H., Corcoran A. F. C., Evans M. J., Colledge W. H., Friedman L. S., Ponder B. A., Venkitaraman A. R. Involvement of Brca2 in DNA repair. Mol. Cell, 1: 347-357, 1998.[Medline]
  13. Paulovich A. G., Toczyski D. P., Hartwell L. H. When checkpoints fail. Cell, 88: 315-321, 1997.[Medline]
  14. Roberts B. T., Farr K. A., Hoyt M. A. The Saccharomyces cerevisiae checkpoint gene BUB1 encodes a novel protein kinase. Mol. Cell. Biol., 14: 8282-8291, 1994.[Abstract/Free Full Text]
  15. Taylor S. S., McKeon F. Kinetochore localization of murine Bub1 is required for normal mitotic timing and checkpoint response to spindle damage. Cell, 89: 727-735, 1997.[Medline]
  16. Cahill D. P., Lengauer C., Yu J., Riggins G. J., Willson J. K., Markowitz S. D., Kinzler K. W., Vogelstein B. Mutations of mitotic checkpoint genes in human cancers. Nature (Lond.), 392: 300-303, 1998.[Medline]
  17. Durfee T., Becherer K., Chen P. L., Yeh S. H., Yang Y., Kilburn A. E., Lee W. H., Elledge S. J. The retinoblastoma protein associates with the protein phosphatase type 1 catalytic subunit. Genes Dev., 7: 555-569, 1993.[Abstract/Free Full Text]
  18. Altschul S. F., Gish W., Miller W., Myers E. W., Lipman D. J. Basic local alignment search tool. J. Mol. Biol., 215: 403-410, 1990.[Medline]
  19. Pearson W. R., Lipman D. J. Improved tools for biological sequence comparison. Proc. Natl. Acad. Sci. USA., 85: 2444-2448, 1988.[Abstract/Free Full Text]
  20. Taylor S. S., Ha E., McKeon F. The human homologue of Bub3 is required for kinetochore localization of Bub1 and a Mad3/Bub1-related protein kinase. J. Cell. Biol., 142: 1-11, 1998.[Abstract/Free Full Text]
  21. Hartwell L. Defects in a cell cycle checkpoint may be responsible for the genomic instability of cancer cells. Cell, 71: 543-546, 1992.[Medline]
  22. Chan G. K. T., Schaar B. T., Yen T. J. Characterization of kinetochore binding domain of CENP-E reveals interactions with the kinetochore proteins CENP-F and BUBR1. J. Cell. Biol., 143: 49-63, 1998.[Abstract/Free Full Text]
  23. Jablonski S. A., Chan G. K., Cooke C. A., Earnshaw W. C., Yen T. J. The hBUB1 and hBUBR1 kinases sequentially assemble onto kinetochores during prophase with hBUBR1 concentrating at the kinetochore plates in mitosis. Chromosoma (Berl.), 107: 386-396, 1998.[Medline]
  24. Chen J., Silver D. P., Walpita D., Cantor S. B., Gazdar A. F., Tomlinson G., Couch F. J., Weber B. L., Ashley T., Livingston D. M., Scully R. Stable interaction between the products of the BRCA1 and BRCA2 tumor suppressor genes in mitotic and meiotic cells. Mol. Cell, 2: 317-328, 1998.[Medline]
  25. Vaughn J. P., Cirisano F. D., Huper G., Berchuck A., Futreal P. A., Marks J. R., Iglehart J. D. Cell cycle control of BRCA2. Cancer Res., 56: 4590-4594, 1996.[Abstract/Free Full Text]
  26. Bertwistle D., Swift S., Marston N. J., Jackson L. E., Crossland S., Crompton M. R., Marshall C. J., Ashworth A. Nuclear location and cell cycle regulation of the BRCA2 protein. Cancer Res., 57: 5485-5488, 1997.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Cancer Res.Home page
J. Wray, J. Liu, J. A. Nickoloff, and Z. Shen
Distinct RAD51 Associations with RAD52 and BCCIP in Response to DNA Damage and Replication Stress
Cancer Res., April 15, 2008; 68(8): 2699 - 2707.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
I. Cousineau, C. Abaji, and A. Belmaaza
BRCA1 Regulates RAD51 Function in Response to DNA Damage and Suppresses Spontaneous Sister Chromatid Replication Slippage: Implications for Sister Chromatid Cohesion, Genome Stability, and Carcinogenesis
Cancer Res., December 15, 2005; 65(24): 11384 - 11391.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. Biol.Home page
H. Lu, X. Guo, X. Meng, J. Liu, C. Allen, J. Wray, J. A. Nickoloff, and Z. Shen
The BRCA2-Interacting Protein BCCIP Functions in RAD51 and BRCA2 Focus Formation and Homologous Recombinational Repair
Mol. Cell. Biol., March 1, 2005; 25(5): 1949 - 1957.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
H.-R. Lin, N. S. Y. Ting, J. Qin, and W.-H. Lee
M Phase-specific Phosphorylation of BRCA2 by Polo-like Kinase 1 Correlates with the Dissociation of the BRCA2-P/CAF Complex
J. Biol. Chem., September 19, 2003; 278(38): 35979 - 35987.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
S. Okada and T. Ouchi
Cell Cycle Differences in DNA Damage-induced BRCA1 Phosphorylation Affect Its Subcellular Localization
J. Biol. Chem., January 10, 2003; 278(3): 2015 - 2020.
[Abstract] [Full Text] [PDF]

Home page
 Hum Mol GenetHome page
M. Warren, A. Smith, N. Partridge, J. Masabanda, D. Griffin, and A. Ashworth
Structural analysis of the chicken BRCA2 gene facilitates identification of functional domains and disease causing mutations
Hum. Mol. Genet., April 1, 2002; 11(7): 841 - 851.
[Abstract] [Full Text] [PDF]

Home page
 J. Cell Sci.Home page
A. R. Venkitaraman
Functions of BRCA1 and BRCA2 in the biological response to DNA damage
J. Cell Sci., March 12, 2002; 114(20): 3591 - 3598.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
Y. Yuan and Z. Shen
Interaction with BRCA2 Suggests a Role for Filamin-1 (hsFLNa) in DNA Damage Response
J. Biol. Chem., December 14, 2001; 276(51): 48318 - 48324.
[Abstract] [Full Text] [PDF]

Home page
 Hum Mol GenetHome page
P. L. Welcsh and M.-C. King
BRCA1 and BRCA2 and the genetics of breast and ovarian cancer
Hum. Mol. Genet., April 1, 2001; 10(7): 705 - 713.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Futamura, M.
Right arrow Articles by Nakamura, Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Futamura, M.
Right arrow Articles by Nakamura, Y.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online