1

BRCA1 Phosphorylation by Aurora-A in the Regulation of G2 to M Transition

Mutsuko Ouchi1, Nobuko Fujiuchi1, Kaori Sasai2, Hiroshi Katayama2,Yohji A. Minamishima4, Pat P. Ongusaha4, Chuxia Deng3, Subrata Sen2, Sam W. Lee4

and Toru Ouchi1*

1 The Derald H. Ruttenberg Cancer CenterThe Mount Sinai School of Medicine

New York UniversityNew York, NY 10029

2 Division of Pathology and Laboratory MedicineThe University of Texas M.D. Anderson Cancer Center

Houston, TX 770303 Genetics of Development and Disease Branch

National Institutes of HealthBethesda, MD 20892

4 Cancer Biology ProgramBeth Israel Deaconess Medical Center and Harvard Medical School

Boston, MA02115

*Corresponding Author:Toru OuchiThe Derald H. Ruttenberg Cancer CenterThe Mount Sinai School of MedicineNew York UniversityBox 1130, One Gustave L. Levy PlaceNew York, NY 10029Tel: (212) 659-5475Fax; (212) 987-2240Email: Toru.Ouchi@mssm.edu

Running Title: Aurora-A Phosphorylates BRCA1

The abbreviations used are:IR: ionizing radiationGy: grayMEFs: mouse embryo fibroblastsATM: Ataxia Telangiectasia Mutatedsi-RNA; small interfering RNAaa; amino acidGST: glutathione S transferase

JBC Papers in Press. Published on February 27, 2004 as Manuscript M311780200

Copyright 2004 by The American Society for Biochemistry and Molecular Biology, Inc.

by guest on Novem

ber 19, 2020http://w

ww

.jbc.org/D

ownloaded from

2

Abstract

Aurora-A/BTAK/STK15 localizes to the centrosome in the G2-M phase and its

kinase activity regulates the G2 to M transition of the cell cycle. Previous studies

have shown that the BRCA1 breast cancer tumor suppressor also localizes to the

centrosome and that BRCA1 inactivation results in loss of the G2-M checkpoint. We

demonstrate here that Aurora-A physically binds to, and phosphorylates, BRCA1.

Biochemical analysis showed that BRCA1 aa1314-1863 binds to Aurora-A. Site-

directed mutagenesis indicated that Ser308 of BRCA1 is phosphorylated by Aurora-

A in vitro. Anti-phospho-specific antibodies against Ser308 of BRCA1 demonstrated

that Ser308 is phosphorylated in vivo. Phosphorylation of Ser308 increased in the

early M phase when Aurora-A activity also increases; these effects could be

abolished by ionizing radiation (IR). Consistent with these observations, acute loss

of Aurora-A by small interfering (si)-RNA resulted in reduced phosphorylation of

BRCA1 Ser308, and transient infection of adenovirus Aurora-A increased Ser308

phosphorylation. Mutation of a single phosphorylation site of BRCA1 (308Ser to Asn,

S308N), when expressed in BRCA1-deficient mouse embryo fibroblasts (MEFs),

decreased the numbers of cells in the M phase to a degree similar to that with wild

type BRCA1-mediated G2 arrest induced by DNA damage. We propose that

BRCA1 phosphorylation by Aurora-A plays a role in G2 to M transition of cell

cycle.

by guest on Novem

ber 19, 2020http://w

ww

.jbc.org/D

ownloaded from

3

Introduction

The breast cancer susceptibility gene, BRCA1, encodes a protein of 1863 amino acids of

nuclear phosphoprotein (1-5). Mutations in the BRCA1 locus have been found in

approximately 40% of familial breast cancers and most of the combined familial breast

and ovarian cancers (6,7). BRCA1 contains a number of structural motifs responsible for

interaction with cellular proteins to regulate diverse biological functions (8,9);

transcription control (10-17), cell cycle regulation (18,19), chromatin remodeling (20-

22), DNA damage repair (23-26) and centrosome localization and duplication (27,28).

Recent studies have demonstrated that phosphorylation of specific residues of BRCA1

protein, such as Ser988, Ser1423 and Ser1524 etc, is important for regulation of survival

of cells with DNA-damage (29-33). Of note, phosphorylation of BRCA1 is induced by

kinases, including Ataxia Telangiectasia Mutated (ATM), ATR, Chk2 and c-Abl, which

are known to be involved in checkpoint machinery. These results indicate that the

physiological function of BRCA1 is at least in part determined by phosphorylation of

specific residues.

The essential functions of BRCA1 have been further explored by BRCA1-null or exon

11-deleted mice (18,28,34-36). Analysis of exon 11-deleted MEFs from such mutant

mice indicates that compared to normal MEFs, which contains one or two centrosomes,

about 25% of mutant MEFs contain more than two, leading to loss of the G2-M

checkpoint and aneuploidy (18). Interestingly, we found that without DNA-damage,

Ser988-phosphorylated BRCA1 resides in the centrosome and chromosome through

metaphase to telophase as a result of the actions of anti-phospho-specific antibodies that

recognize phosphorylated Ser988 of BRCA1 (5). These results reinforce the working

by guest on Novem

ber 19, 2020http://w

ww

.jbc.org/D

ownloaded from

4

hypothesis that BRCA1 plays an important role in mitosis, but the role of

phosphorylation of the protein in regulation of the M phase is largely unknown.

The Aurora-A gene locus is located in the 20q13 chromosome region, which is frequently

amplified in several different types of malignant tumors such as breast, colorectal,

pancreatic and bladder cancers (37,38). In particular, the 20q11-q13 region is amplified

in 40% of breast cancer cell lines as well as in 12% to 18% of primary tumors.

The encoded protein, Aurora-A, is a member of the Ser/Thr kinase family, and recent

studies have demonstrated that Aurora-A is involved in the G2-M checkpoint and mitosis

commitment (39-42). In Drosophila, mutations in the aurora-A gene result in incomplete

centrosome positioning, leading to abnormal formation of spindle poles and astral

microtubules (43,44). In Xenopus egg extracts, Eg2, a homologue of Aurora-A, has been

shown to be involved in mitotic spindle assembly (45). Furthermore, recent studies have

demonstrated that Aurora-A is inactivated by DNA damage at the end of the G2 phase

and over-expression of Aurora-A abrogates the G2 checkpoint in higher eukaryotes (39).

Significantly, Aurora-A is recruited to the centrosome during early G2 and it becomes

phosphorylated and activated in centrosomes late in the G2 phase (42). Moreover, small

interfering (si) RNA-mediated depletion of Aurora-A results in the failure to enter

mitosis, suggesting that Aurora-A dependent signaling in the centrosome is crucial for

mitosis commitment.

On the basis of these observations, we explored whether BRCA1 regulates mitosis entry

through functional interaction with Aurora-A in the G2 to M phases. Our results

demonstrate that Aurora-A binds to BRCA1 and phosphorylates Ser308 of BRCA1. A

phospho-specific antibody recognizing phosphorylated Ser308 showed that this

by guest on Novem

ber 19, 2020http://w

ww

.jbc.org/D

ownloaded from

5

phosphorylation increases in early M phase but is inhibited by IR damage. A biological

assay by means of BRCA1 (-/-) MEFs revealed that BRCA1 phosphorylation by Aurora-

A might regulate mitotic entry. Given that both Aurora-A and BRCA1 are closely

associated with breast carcinogenesis, our results suggest a model in which derailed

regulation of G2-M transition by these proteins predisposes to cancer development.

by guest on Novem

ber 19, 2020http://w

ww

.jbc.org/D

ownloaded from

6

Experimental Procedures

Cell Culture, Synchronization and Fluorescence-activated Cell Sorter Analysis

MCF7, U2OS cells and BRCA1 (-/-) MEFs (46) were maintained in Dulbecco’s modified

Eagle’s medium (DMEM)/10% fetal bovine serum (FBS). For synchronization of the cell

cycle at the G1/S boundary, a double-thymidine block and release was performed (47).

Briefly, MCF7 cells were treated with 2.5mM thymidine for 16h and then released by

washing them with phosphate-buffered saline (PBS) three times followed by placing

them in fresh medium containing DMEM/10% FBS. After 8h, cells were re-treated with

thymidine for 16h. Cells were washed with PBS again and maintained in fresh

DMEM/10% FBS medium containing 20nM roscovitine (Sigma) for 12h to synchronize

them at the G2 phase. The cells were washed with PBS to release them into mitosis, and

M phase cells were collected after 30min. For each time point, cells were stained with

propidium iodide and FITC-conjugated anti-phospho-histone H3 antibody (Cell

Signaling) as described previously (48). IR was administered using a MARK2

IRRADIATOR (J. L. Shepherd & Associates, San Fernando, CA).

Plasmid Construction and Adenovirus Production

Wild type and kinase-defective forms of Aurora-A have been described previously (49).

BRCA1Ser308Asn was generated using QuickChange (Stratagene) with primers of 5’-

TGTAATAAAAACAAACAGCCT-3’ and its complement sequence. FLAG-tagged

BRCA1 cDNA was subcloned into pBabepuro and retrovirus was generated as described

(50). Both cDNAs were subcloned into a pAdTRACK-CMV vector and recombination

was performed in a BJ5180 bacterial strain as reported previously (11). Mammalian

by guest on Novem

ber 19, 2020http://w

ww

.jbc.org/D

ownloaded from

7

GST-tag vector, pEBG, was obtained from Bruce Mayer at the University of Connecticut

Health Center.

Transfection, Immunoprecipitation and Immunoblot Analysis

For si-RNA analysis of Aurora-A, double strand RNA (5’-AAAUGCCCUGUCUUACU

GUCA-3’) was synthesized (Dharmacon) and transfected with OligofectAMINE

(Invitrogen). Transfection was performed with Fugene (Roche) or LipofectAMINE (Life

Technologies, Inc.) according to the manufacturer’s protocol. Rabbit polyclonal phospho-

Ser-specific antibody recognizing phosphorylated Ser308 of BRCA1 was generated by

Research Genetics, Inc. against KLH-conjugated synthetic peptides;

EFCNKSpKQPGLAR. The following antibodies were purchased for western blot

analysis: antibodies for BRCA1 (C-20, Santa Cruz; Ab-1, Calbiochem), Aurora-A

(#3092, Cell Signaling), Aurora-A/T288-P (#3091, Cell Signaling), GST (Z-5, Santa

Cruz), Actin (H196, Santa Cruz) and FLAG (M2, Sigma)). Cell extracts were prepared in

EBC buffer (50mM HEPES, pH 7.6, 250mM NaCl, 0.1% NP-40, 5mM EDTA, pH 8.0,

with mixed protease inhibitor (Sigma). Total cell lysates (1mg) were used for

immunoprecipitation with the indicated antibodies and Protein G-agarose (Sigma).

Samples were washed with EBC buffer four times and subjected to SDS-PAGE. The

secondary antibodies (Jackson Immunolaboratory) were peroxidase-conjugated anti-

mouse IgG (H+L) or anti-rabbit IgG (H+L). Films were developed by ECL.

GST Pull-Down Assay

by guest on Novem

ber 19, 2020http://w

ww

.jbc.org/D

ownloaded from

8

Purification of GST-BRCA1 constructs was described previously (25). Purified Aurora-

A was produced by cleavage of GST-Aurora-A by thrombin according to the

manufacturers protocol (Amersham Biosciences). Briefly, 1µg of purified Aurora-A was

incubated with 1µg of GST-BRCA1 fusion proteins for 1h at 4°C. Glutathione beads

were added to each sample, and samples were further rotated for 1h at 4°C. After

extensive washing with NET-N buffer (20mM Tris-HCl, pH 8.0, 100mM NaCl, 1mM

EDTA, 0.5% NP-40, 100mM NaF, 200µM sodium orthovanadate) followed by NET-N

buffer containing 500mM of NaCl, samples were loaded in 7.5% SDS-PAGE and blotted

with anti Aurora-A antibody.

GST pull-down assay using total cell lysates of MCF7 cells were described previously

(10,11). Briefly, 500µg of lysates were incubated with about 1µg of GST-fusion proteins

for 1h at 4°C. After glutathione beads were added to each sample, samples were further

rotated for 1h at 4°C. After extensive washing with NET-N buffer, samples were loaded

in 7.5% SDS-PAGE and blotted with anti Aurora-A antibody.

In Vitro Kinase Assay

Immunoprecipitates were washed with NET-N buffer containing 500mM of NaCl

followed by kinase buffer (50mM Tris-HCl, pH7.5, 15mM MgCl2, 1mM dithiothreitol).

Each sample was incubated with 20µl of kinase buffer containing 5µCi of [γ-32P] ATP

and 1µg of GST-BRCA1 proteins for 10min at 30oC and then separated in SDS-PAGE.

Gels were dried and autoradiographed.

by guest on Novem

ber 19, 2020http://w

ww

.jbc.org/D

ownloaded from

9

Results

BRCA1 Forms a Complex with Aurora-A

Aurora-A/BTAK/STK15 is a Ser/Thr kinase, which is localized in the centrosome and

frequently amplified in human cancer (38-42). Recent studies have shown that Aurora-A

plays a crucial role in mitotic entry and that Aurora-A activation in the G2-M transition is

inhibited by DNA damage (39). Since we and others have found that BRCA1 is also

localized in the centrosome (5,27), we examined whether BRCA1 and Aurora-A form a

complex in normally growing cells. BRCA1 was immunoprecipitated from

unsynchronized BRCA1-mutated HCC1937 or MCF7 cells with anti-BRCA1 antibodies,

C-20 and Ab-1 as indicated in Figure 1A. Samples were immunoblotted with anti-

Aurora-A antibody. As shown in Figure 1A, Aurora-A was detected in the BRCA1

immunoprecipitates from MCF7 cells.

We next determined the Aurora-A binding region of BRCA1. BRCA1 aa1-324, aa260-

553, aa502-802, aa758-1064, aa1005-1313 and aa1314-1863 were expressed as N-

terminal GST fusion proteins in bacteria. After purification, the GST-fusion proteins

were incubated with purified Aurora-A protein and samples were extensively washed by

NET-N buffer containing 500mM of NaCl as described in Experimental Procedures

followed by immunoblotted with anti-Aurora-A antibody. As shown in Figure 1B top,

interaction with Aurora-A was detected in aa1314-1863 region. Interestingly, when cell

lysates were incubated with six GST-BRCA1 fragments above, both aa758-1064 and

aa1314-1863 segments of BRCA1 were found to bind to Aurora-A (Figure 1B, bottom).

These results suggest that, although BRCA1 aa1314-1863 is a primary binding region to

Aurora-A, BRCA1 aa758-1064 may indirectly bind to Aurora-A through cellular

by guest on Novem

ber 19, 2020http://w

ww

.jbc.org/D

ownloaded from

10

protein(s). GST-BRCA1 fragments were immunoblotted in the separate SDS-PAGE with

anti-GST antibody to confirm that similar amounts of GST-fusion proteins were used for

this assay (Figure 1C).

These results demonstrate that BRCA1 forms a complex with Aurora-A in vivo.

Aurora-A Phosphorylates BRCA1 in vitro

Since both BRCA1 and Aurora-A coexist in the centrosome and form a complex in vivo

as shown above, we investigated whether BRCA1 is phosphorylated by Aurora-A. GST-

tagged Aurora-A was expressed in 293T cells and affinity-precipitated with GSH

(glutathione)-beads. Samples were extensively washed with NET-N buffer containing

500mM of NaCl and divided into six aliquots and an in vitro kinase assay performed

using as a substrate the purified GST-fusion proteins of BRCA1 from the experiment

shown in Figure 1C. Expression of GST-tagged Aurora-A was confirmed by anti-GST

immunoblot analysis after GST-pull down of the protein with GSH beads (Figure 2A). As

shown in Figure 2B, GST-Aurora-A strongly phosphorylated BRCA1 aa260-553 and,

less efficiently, the aa1314-1863 segments. To rule out the possibility that an unknown

kinase(s) binding to the GST region of GST-Aurora-A phosphorylated the substrates, the

same in vitro kinase assay was performed expressing GST protein alone in 293T cells

followed by affinity-purification. Since precipitated GST did not phosphorylate BRCA1

segments used in the experiment shown in Figure 2B, phosphorylation of BRCA1 aa260-

553 is not carried out by a kinase(s) that binds to the GST tag (data not shown). We

confirmed that similar levels of GST-BRCA1 proteins were used as substrates by

coomassie blue-staining of a gel (Figure 2C). Aurora-A-dependent phosphorylation of

by guest on Novem

ber 19, 2020http://w

ww

.jbc.org/D

ownloaded from

11

BRCA1 was further confirmed by means of a kinase-deficient (KD) form of Aurora-A, in

which lysine162 was mutated to arginine (49); Wild type (WT) or KD forms of Aurora-A

with the N-terminal GST tag was expressed in 293T cells and affinity-precipitated with

GSH-beads as described in Figure 2A. After extensive washing of the samples with NET-

N buffer containing high salt concentration, in vitro kinase assay demonstrated that

purified GST-BRCA1(260-553) was phosphorylated by the WT of Aurora-A, not by the

KD form, showing that BRCA1(260-553) is phosphorylated by Aurora-A. These results

demonstrate that BRCA1 is a substrate of Aurora-A in vitro.

Aurora-A Phosphorylates Ser308 of BRCA1

Previous studies identified putative consensus phosphorylation sites of Aurora-A using

mass spectrometry analysis of 28 kinetochore proteins phosphorylated by Aurora-A (51).

Among these motifs are K{S/T} sequences whose phosphorylation by lpl1p/Aurora-A

have been found in Dam1, Spc34 and the autophosphorylation site of lpl1p/Aurora. In

BRCA1 aa260-553, five Ser and Thr residues fit this motif (Ser308, Ser444, Ser451,

Thr464 and Thr528), and mutagenic analysis (S308N, S444A, S451A, T464A and

T528A) revealed that phosphorylation of BRCA1 aa260-553 by Aurora-A is abolished

only in S308N mutant (Figure 3A and data not shown).

We generated rabbit anti-phospho-specific antibody, S308P, which recognizes

phosphorylated Ser308 of BRCA1 protein, and further studied in vivo phosphorylation of

the protein. To characterize S308P antibody, total cell lysates of MCF7 cells were treated

by λ-phosphatase and immunoblotted with anti BRCA1 or S308 antibodies. After

treatment, shift-down of the protein was detected by an anti-BRCA1 antibody

by guest on Novem

ber 19, 2020http://w

ww

.jbc.org/D

ownloaded from

12

(MAb21A8, ref. 5). S308 did not recognize the protein after phosphatase treatment,

indicating that the S308 antibody specifically detects the Ser308-phosphorylated form of

BRCA1 (Figure 3B).

We further explored the phosphorylation of Ser308 in transition from the G2 to the M

phase under conditions of DNA damage. The cell cycle of MCF7 cells was synchronized

at the G2 phase by a double-thymidine-block followed by roscovitine treatment (see

EXPERIMENTAL PROCEDURES). Briefly, S phase cells synchronized by double-

thymidine-block treatment were washed with PBS and incubated with fresh medium

containing 20nM roscovitine for 12h to arrest at the G2 phase. Rapid assessment of the

progression of cells from the G2 to the M phase is difficult because both G2 and cells

undergoing mitosis contain a 4N DNA content and thus are not distinguishable from one

another by standard propidium iodide (PI) staining and flow cytometry. Since histone H3

is phosphorylated exclusively during mitosis, an antibody that specifically recognizes the

phosphorylated form of histone H3 can be used to identify the cells in mitosis and thus

distinguish them from the G2 cells by flow cytometry (52, 53). Co-staining of cells with

PI to assess DNA content and an anti-phospho-histone H3 antibody demonstrates that the

cells in mitosis can be distinguished from G2 cells in the 4N population of cells. We used

anti-phospho-histone H3 antibody to quantify the mitosis population (Figure 3C). We

found that unsynchronized MCF7 cells contained 1.64% of cells in mitosis, and that this

number was decreased when cells were arrested at the G2 phase (0.24%). As predicted,

the numbers of cells in mitosis increased when cells were released from G2 arrest

(2.99%). Using these protocols to synchronize the cells, we further studied

phosphorylation of Ser308 of BRCA1 in the G2 and M phases. To study whether

by guest on Novem

ber 19, 2020http://w

ww

.jbc.org/D

ownloaded from

13

phosphorylation of Ser308 is affected by DNA damage, cells were treated with IR (5Gy)

immediately after they were released from G2 arrest. Cell lysates were prepared at 1h, 2h

and 3h after the cells were released to entry into mitosis. Immunoblot analysis

demonstrated that phosphorylation of Ser308 is prominent in the M phase gradually

decreased in 2h to 3h after entry into mitosis. Significantly, Ser308 phosphorylation was

severely inhibited when cells were exposed to IR damage at the entry into M phase.

It has been shown that both protein levels and kinase activity of Aurora-A increase during

the late G2 to M phase (39,42). Phosphorylation of Thr288 of Aurora-A, which is located

in the T loop of the kinase domain, results in a marked increase in enzymatic activity

(52). Using the cell lysates of Figure 3D, we studied whether Aurora-A activity is down-

regulated in IR-treated cells (Figure 3E). Consistent with previous studies, protein levels

of Aurora-A significantly increased in M phase. Phosphorylation of Thr288 also

increased in the M phase, suggesting that kinase activity also increased in this phase.

Under IR damage, phosphorylation of Thr288 was severely inhibited, whereas increase in

protein level was weakly decreased. Taken together, these results demonstrate that

Aurora-A phosphorylates Ser308 of BRCA1 when cells enter mitosis, and this

phosphorylation is inhibited under DNA damage as a result of inhibition of Aurora-A

activation.

Decreasing Aurora-A Protein by si-RNA Results in Reduced Ser308

Phosphorylation

Aurora-A-dependent phosphorylation of Ser308 of BRCA1 was further investigated by

decreasing the endogenous protein levels of Aurora-A with small interfering (si)-RNA.

by guest on Novem

ber 19, 2020http://w

ww

.jbc.org/D

ownloaded from

14

We used human osteosarcoma cell line U2OS because these cells are highly

transfectable. U2OS cells were transfected with synthesized double strand RNA specific

for the Aurora-A sequence. After 18h, cell lysates were immunoblotted as indicated in

Figure 4A. Decreased levels of Aurora-A protein levels did not affect the levels of

BRCA1 protein, but phosphorylation of Ser308 was markedly reduced in Aurora-A

knock-down cells. These results reinforce the hypothesis that Aurora-A phosphorylates

Ser308 of BRCA1 in vivo.

Transient Expression of Aurora-A, but Not the Kinase-Defective Form, Induces

Ser308 Phosphorylation

We generated recombinant adenovirus expressing wild type- or kinase-defective (KD,

K162R)-forms of Aurora-A (49), and Ser308 phosphorylation of BRCA1 was examined

by infection with MCF7 cells. Normally growing MCF7 cells were infected by Aurora-A

virus (Ad-Aurora-A, Ad-Aurora-A-KD) or control LacZ virus (Ad-LacZ) and cell lysates

were collected 24h after infection. Total cell lysates were immunoblotted with anti

BRCA1, S308P, anti Aurora-A and anti actin antibodies. As shown in Figure 4B,

transient expression of wild type Aurora-A, but not the KD form, increased Ser308

phosphorylation. These results further demonstrate that Aurora-A phosphorylates Ser308

of BRCA1 protein.

BRCA1 Ser308 Phosphorylation by Aurora-A Influences G2 to M Progression

The results presented thus far demonstrate that BRCA1 becomes phosphorylated by

Aurora-A when cells enter mitosis. We explored the role of Ser308 phosphorylation in

by guest on Novem

ber 19, 2020http://w

ww

.jbc.org/D

ownloaded from

15

G2-M progression. BRCA1(-/-) MEFs were obtained from BRCA1 exon 11-deleted mice

(46) and infected for 48h with retrovirus expressing wild type (WT)- or the S308N form

of BRCA1 with an N-terminal FLAG tag. Normal MEFs were also infected with

retrovirus expressing S308N form of BRCA1. A LacZ virus was used as a control.

Expression of BRCA1 was confirmed by immunoblot analysis with anti FLAG antibody

(Figure 5A). The numbers of cells in the M phase were quantified by means of anti-

phospho-specific histone H3 antibody as described in Figure 3. As shown in Figure 5B,

BRCA1(-/-) MEFs expressing WT showed a similar population of M phase (2.02%) with

LacZ cells (2.23%). Treatment of BRCA1(-/-) cells expressing WT with IR (10Gy)

resulted in significant reduction in the number of M phase (0.35%), presumably as a

result of activation of G2-M checkpoint, which prevented cells from entering mitosis.

Importantly, reexpression of the S308N mutant form of BRCA1 in both BRCA1(+/+) and

(-/-) MEFs showed reduction of the number of M phase without DNA damage (0.52%

and 0.47%, respectively), a level similar to those in BRCA1(-/-) expressing WT treated

with IR. Although IR treatment did not affect the number of M phase of BRCA1(-/-) cells

infected with LacZ (2.30%), BRCA1(-/-) cells expressing the S308N BRCA1 show the

slightly lower numbers of M phase compared to BRCA1(-/-) cells expressing WT under

these conditions (0.23% and 0.35%, respectively). Significant apoptosis was not detected

in these conditions and BRCA1(+/+) MEFs infected with LacZ or WT did not show the

reduced levels of M phase (data not shown). These results demonstrate that cells

expressing non-phosphorylated form of BRCA1 at Ser308 do not proceed from G2 to M

phase.

by guest on Novem

ber 19, 2020http://w

ww

.jbc.org/D

ownloaded from

16

Discussion

We and others have demonstrated BRCA1 localization in the centrosome during the M

phase (5,27). This observation was confirmed by coimmunostaining of different types of

cell lines with antibodies to γ-tubulin and pericentrin, both well-characterized

components of the centrosome. Consistent with this, biochemical analysis has

demonstrated that BRCA1 aa504-803 is associated with γ-tubulin isolated from cells

arrested at the G2/M phase (27). The biological roles of BRCA1-interaction with γ-

tubulin have been further studied by expressing exon 11 of BRCA1, which contains the γ-

tubulin binding region; ectopic expression of BRCA1 exon 11 in transfected cells caused

centrosome amplification and mitotic abnormalities similarly observed in BRCA1 exon

11-deleted MEFs (46). These results strongly suggest that BRCA1 plays a crucial role in

regulating the G-M progression; however the molecular pathway of BRCA1 regulation

underlying the G2-M progression is not clear.

Recent studies have shown that Aurora-A activity, in conjunction with LIM protein

Ajuba, is required for entry into mitosis (42). These studies have also shown that initial

Aurora-A activation occurs in the late G2 phase of the cell cycle and is required for

recruitment of the cyclin B1-cdk1 complex to the centrosome. Significantly, activation of

Aurora-A at the G2-M transition was inhibited when cells were exposed to adriamycin at

the S phase (39), and our current results also demonstrate that IR treatment leads to an

inhibition of phosphorylation of Aurora-A Thr288, which is required for maximum

kinase activation (52). These studies reinforce a model in which the kinase activity of

by guest on Novem

ber 19, 2020http://w

ww

.jbc.org/D

ownloaded from

17

Aurora-A positively regulates the G2 to M transition; however downstream targets of the

kinase in this pathway remain unclear.

On the basis of these observations, we investigated whether BRCA1 is involved in

Aurora-A-mediated regulation of the G2-M phase. Our results showing

coimmunoprecipitation of both proteins demonstrate that BRCA1 and Aurora-A form a

complex in vivo. Since BRCA1 localizes in the centrosome in the M phase (5,27) and

Aurora-A is detected only on duplicated centrosomes from the end of the S phase to the

beginning of the G1 phase of the next cycle (50), it is likely that this complex exists from

the late G2 to the M phase.

Biochemical analyses have identified several motifs that can be phosphorylated by

Aurora-A (51,54). Among them are the K{S,T} motif, whose phosphorylation by Aurora-

A was detected in Dam1, Spc34 and the lpl1p/Aurora autophosphorylation site and was

confirmed in vivo by mass spectrometry (50). We identified Ser308 as a major

phosphorylation site of the BRCA1 aa260-553 region by Aurora-A, which is efficiently

phosphorylated by an in vitro kinase assay. Since this region of BRCA1 contains five

Ser/Thr residues that fit this motif (Ser308, Ser444, Ser451, Thr464 and Thr528), optimal

phosphorylation of Ser308 is probably determined by the adjacent amino acid sequence.

It has been shown that MEFs carrying mutant alleles of BRCA1 contain more than two

centrosomes compared to normal MEFs containing one or two. Also, centrosome

maturation from the late S to the M phase is critical for completion of mitosis (56). One

may speculate that immature chromosome segregation due to lack of functional

by guest on Novem

ber 19, 2020http://w

ww

.jbc.org/D

ownloaded from

18

centrosomes causes multinucleation and multicentrosomes, which are the hallmark of

cancer cells. Identification of proteins that cooperate with BRCA1 in mitosis needs to be

extended to further understand the mechanism by which loss of BRCA1 establishes a

predisposition to cancer.

by guest on Novem

ber 19, 2020http://w

ww

.jbc.org/D

ownloaded from

19

Acknowledgement

We are grateful to members of the Ouchi laboratory for critical discussion. This work

was supported by an award from the New York City Council’s Speaker’s Fund (T.O.), a

State of New York EMPIRE Grant (T.O.) and the National Cancer Institute (CA79892

and CA90631 to T.O.).

by guest on Novem

ber 19, 2020http://w

ww

.jbc.org/D

ownloaded from

20

References

1 . Miki, Y., Swensen, J., Shattuck-Eidens, D., Futreal, P.A., Harshman, K.,

Tavtigian, S., Liu, Q., Cochran, C., Bennet, L.M., Ding, W., et al. (1994) Science

266, 66-71

2. Thomas, J.E., Smith, M., Rubinfeld, B., Gutowski, M., Beckmann, R.P. and

Polakis, P. (1996) J. Biol. Chem. 271, 28630-28635

3. Scully, R., Chen, J., Ochs, R., Keegan, K., Hoekstra, M., Feunteun, J. and

Livingston, D.M. (1997) Cell 90, 425-435

4. Chen, Y., Chen, C.-H., Riley, D.J., Allred, C., Chen, P.-L., von Hoff, D.V.,

Osborne, C.K. and Lee, W.-H. (1995) Science 270, 789-791

5. Okada, S. and Ouchi, T. (2003) J. Bio. Chem. 278, 2015-2020

6. Alberg, A.J. and Helzlsouer, K.J. (1997) Curr. Opin. Oncol. 9, 505-511

7. Alberg, A.J., Lam, A.P. and Helslsouer, K.J. (1999) Curr. Opin. Oncol. 11, 435-

441

8. Deng, C. and Brodie, S.G. (2000) Bioessays 22. 728-737

9. Rosen, E.M., Fan, S., Pestell, R.G. and Goldberg, I.D. (2003) J. Cell. Physiol.

196, 19-41

10. Ouchi, T., Monteiro, A.N., August, A., Aaronson, S.A. and Hanafusa, H. (1998)

Proc. Natl. Acad. Sci. USA. 95, 2302-2306

11. Ouchi, T., Lee, S.W., Ouchi, M., Aaronson, S.A. and Horvath, C.M. (2000) Proc,

Natl. Acad. Sci. USA. 97, 5208-5213

12. Aprelikova, O., Pace, A.J., Fang, B., Koller, B.H. and Liu, E.T. (2001) J. Biol.

Chem. 276, 25647-25650

by guest on Novem

ber 19, 2020http://w

ww

.jbc.org/D

ownloaded from

21

13. Chai, Y.L., Chi, J., Shao, N., Shyam, E., Reddy, P. and Rao, V.N. (1999)

Oncogene 18, 263-268

14. Chapman, M.S. and Verma, I.M. (1996) Nature 382, 678-679

15. Fan, S., Wang, J., Yuan, R., Ma, Y., Meng, Q., Erdos, M.R., Pestell, R.G., Yuan,

F., Auborn, K.J., Goldberg, I.D. and Rosen, E.M. (1999) Science 284, 1354-1356

16. Zhang, H., Somasundaram, K., Peng, Y., Tian, H., Bi, D., Weber, B.L. and El-

Deiry W.S. (1998) Oncogene 16, 1713-1721

17. Zheng, L., Annab, L.A., Afshari, C.A., Lee, W.-H. and Boyer, T.G. (2001) Proc.

Natl. Acad. Sci. USA. 98, 9587-9592

18. Xu, X., Weaver, Z., Linke, S.P., Li, C., Gotay, J., Wang, X.W., Harris, C.C.,

Ried, T. and Deng, C.X. (1999 ) Mol. Cell 3, 389-395

19. Yarden, R.I., Pardo-Reoyo, S., Sgagias, M., Cowan, K.H. and Brody, L.C. (2002)

Nat. Genet. 30, 285-289

20. Bochar, D.A., Wang, L., Beniya, H., Kinev, A., Xue, Y., Lane, W.S., Wang, W.,

Kashanchi, F. and Shiekhattar, R. (2000) Cell 102, 257-265

21. Hu, Y.F., Hao, Z.L. and Li, R. (1999) Genes Dev. 13, 637-642

22. Ye, Q., Hu, Y.F., Zhong, H., Nye, A.C., Belmont, A.S. and Li, R. (2001) J. Cell.

Biol. 155, 911-921

23. Gowen, L.C., Avrutskaya, A.V., Latour, A.M., Koller, B.H. and Leadon, S.A.

(1998) Science 281, 1009-1012

24. Moynahan, M.E., Chiu, J.W., Koller, B.H. and Jasin, M. (1999) Mol. Cell 4, 511-

518

by guest on Novem

ber 19, 2020http://w

ww

.jbc.org/D

ownloaded from

22

25. Scully, R., Chen, J., Plug, A., Xiao, Y., Weaver, D., Feunteun, J., Ashley, T. and

Livingston, D.M. (1997) Cell 88, 265-275

26. Zhong, Q., Chen, C.F., Le, S., Chen, Y., Wang, C.C., Xiao, J., Chen, P.L., Sharp,

Z.D. and lee, W.-H. (1999) Science 285, 747-750

27. Hsu, L.C., Doan, T.P. and White, R.L. (2001) Cancer Res. 61, 7713-7718

28. Xu, X., Wagner, K.U., Larson, D., Weaver, Z., Li, C., Ried, T., Hennighausen, L.,

Wynshaw-Boris, A. and Deng, C.X. (1999) Nat. Genet. 22, 37-43

29. Gatei, M., Zhou, B.-B., Hobson, K., Scott, S., Young, D. and Khanna, K.K.

(2001) J. Biol. Chem. 276, 17276-17280

30. Xu, B., O’Donnell, A.H., Kim, S.-T. and Kastan, M.B. (2002) Cancer Res. 62,

4588-4591

31. Cortez, D., Wang, Y., Qin, J. and Elledge, S.J. (1999) Science 286, 1162-1166

32. Tibbetts, R.S., Cortez, D., Brumbaugh, K.M., Scully, R., Livingston, D., Elledge,

S.J. and Abraham, R.T. (2000) Genes Dev. 14, 2989-3002

33. Foray, N., Marot, D., Randrianarison, V., Venezia, N.D., Picard, D., Perricaudet,

M., Favaudon, V. and Jeggo, P. (2002) Mol. Cell. Biol. 22, 4020-4032

34. Hakem, R.,. de la Pompa, J.L., Sirard, C., Mo, R., Woo, M., Hakem, A.,

Wakeham, A., Potter, J., Reitmair, A., Billia, F., Firpo, E., Hui, C.C., Roberts, J.,

Rossant, J. and Mak, T.W. (1996) Cell 85, 1009-1023

35. Liu, C.-Y., Flesken-Nikitin, A., Li, S., Zeng, Y. and lee, W.-H. (1996) Genes

Dev. 10, 1835-1843

36. Gowen, L.C., Johnson, B.L., Latour, A.M., Sulik, K.K. and Koller, B.H. (1996)

Nat. Genet. 12, 191-194

by guest on Novem

ber 19, 2020http://w

ww

.jbc.org/D

ownloaded from

23

37. Bischoff, J.R., Anderson, L., Zhu, Y., Mossie, K., Ng, L., Souza, B., Schryver, B.,

Flanagan, P., Clairvoyant, F., Ginther, C., Chan, C.S., Novotny, M., Slamon, D.J.

and Plowman, G.D. (1998) EMBO J. 17, 3052-3065

38. Zhou, H., Kuang, J., Zhong, L., Kuo, W.L., Gray, J.W., Sahin, A., Brinkley, B.R.

and Sen, S. (1998) Nat. Genet., 20, 189-193

39. Marumoto, T., Hirota, T., Morisaki, T., Kunitoku, N., Zhang, D., Ichikawa, Y.,

Sasayama, T., Kuninaka, S., Mimori, T., Tamaki, N.., Kimura, M.., Okano, Y.

and Saya, H. (2002) Genes Cells 7, 1173-1182

40. Meraldi, P., Honda, R. and Nigg, E. (2002) EMBO J. 21, 483-492

41. Anand, S., Penrhyn-Lowe, S. and Venkitaraman, A.R. (2003) Cancer Cell 3, 51-

62

42. Hirota, T., Kunitoku, N., Sasayama, T., Marumoto, T., Zhang, D.., Nitta, M.,

Hatakeyama, K. and Saya, H. (2003) Cell 114, 585-598

43. Giet, R., McLean, D., Descamps, S., Lee, M.J., Raff, J.W., Prigent, C. and

Glover, D.M. (2002) J. Cell Biol. 156, 437-451

44. Glover, D.M., Leibovitz, M.H., McLean, D.A. and Parry, H. (1995) Cell 81, 95-

105

45. Roghi, C., Giet, R., Uzbekov, R., Morin, N., Chartrain, I., Le Guellec, R.,

Couturier, A., Doree, M., Philippe, M. and Prigent, C. (1998) J. Cell Sci. 111,

557-572

46. Xu, X., Qiao, W., Linke, S.P., Cao, L., Li, W.-M., Furth, P.A., Harris, C.C. and

Deng, C.X. (2001) Nat. Genet. 28, 266-271

by guest on Novem

ber 19, 2020http://w

ww

.jbc.org/D

ownloaded from

24

47. Spector, D.L., Goldman, R.D. and Leinwand, L.A. (1998) Cells, a laboratory

manual, Cold Spring Harbor Press.

48. Xu, B., Kim, S.-T. and Kastan, M.B. (2001) Mol. Cell. Biol. 21, 3445-3450

49. Katayama, H., Zhou, H., Li, Q., Tatsuka, M. and Sen, S. (2001) J. Biol. Chem.

276, 46219-46224

50. Aglipay, J.A., Lee, S.W., Okada, S., Fujiuchi, N., Phtsuka, T., Wang, Y.,

Johnstone, R.W., Deng, C.X., Qin, J. and Ouchi, T. (2003) Oncogene, in press.

51. Cheeseman, I.M., Anderson, S., Jwa, M., Green, E.M., Kang, J.-S., Yates III, J.R.,

Chan, C.S.M., Drubin, D.G. and Barnes, G. (2002) Cell 111, 163-172

52. Juan, G., Traganos, F., James, W., Ray, J., Roberge, M., Sauve, D., Anderson, H.

and Darzynkiewicz, Z. (1998) Cytometry 32, 71-77

53. Walter, A.O., Seghezzi, W., Korver, W., Sheung, J. and Lees, E. (2000)

Oncogene 19, 4906-4916

54. Mendez, R., Hake, L.E., Andresson, T., Littlepage, L.E., Ruderman, J.V. and

Richter, J.D. (2000) Nature 404, 302-307

55. Ruffner, H., jiang, W., Craig, A.G., Hunter, T. and Verma, I.M. (1999) Mol. Cell.

Biol. 19, 4843-4854

56. Nigg, E.A. (2002) Nat. Rev. Cancer 2, 1-11

by guest on Novem

ber 19, 2020http://w

ww

.jbc.org/D

ownloaded from

25

Figure Legends

FIG.1 BRCA1 coimmunoprecipitates Aurora-A.

A . Total cell lysates (1mg) of HCC1937 or MCF7 cells were used for

immunoprecipitation (IP) with control rabbit IgG or anti-BRCA1 antibodies (C-

20 and Ab-1). Samples were separated in 7.5% SDS-PAGE and immunoblotted

with anti-Aurora-A antibody. Twenty micrograms of lysates were loaded as a

control.

B. (top) Purified Aurora-A produced in bacteria was incubated with purified GST-

BRCA1 fragments to determine the Aurora-A binding region of BRCA1. (bottom)

Total cell lysates of MCF7 cells were used to determine the Aurora-A binding

region of BRCA1 following the previously described protocol (10,11). Samples

were incubated with glutathione beads, subjected to 7.5% SDS-PAGE, and

immunoblotted with anti-Aurora-A antibody.

C. BRCA1 GST-fusion proteins used for B was confirmed in a separate immunoblot

probed with anti-GST antibody.

FIG. 2 Aurora-A phosphorylates the BRCA1 aa260-553 region in vitro.

A . GST alone (pEBG vector) or GST-tagged Aurora-A (Aurora-A/pEBG) was

transiently expressed in 293T cells and precipitated with GSH beads. Samples

were loaded in 7.5% SDS-PAGE and immunoblotted with anti GST antibody.

The arrow indicates GST-Aurora-A.

by guest on Novem

ber 19, 2020http://w

ww

.jbc.org/D

ownloaded from

26

B. GST-pull down samples from A were divided into six aliquots and incubated for

10min with 1µg of the purified GST-BRCA1 proteins indicated in kinase buffer

containing [γ-32P] ATP. Samples were separated in 7.5% SDS-PAGE and

autoradiographed.

C. A SDS-PAGE gel used in B was stained by Coomasie blue to confirm the amount

of GST-fusion protein used in B.

D. GST alone (pEBG vector), GST-tagged wild type- (WT) or kinase-deficient (KD)

forms of Aurora-A were transiently expressed in 293T cells. After affinity-

precipitation with GSH beads, samples were extensively washed with NET-N

buffer containing 500mM of NaCl and in vitro kinase assay was performed using

GST-BRCA1(260-553) as a substrate.

FIG. 3 Aurora-A phosphorylates Ser308 of BRCA1.

A. After GST-pull down of GST-Aurora-A as described in the FIG. 2 caption, an in

vitro kinase assay was performed using GST-BRCA1 aa260-553 or GST-BRCA1

aa260-553 carrying the 308Ser to Asn mutation as a substrate.

B . Characterization of phospho-specific antibody S308-P recognizing Ser308-

phosphorylated BRCA1. Total cell lysates of MCF7 was treated with λ-

phosphatase and immunoblotted with C-20 or S308-P antibodies.

C. Synchronization of the cell cycle by a double-thymidine block. Unsynchronized

MCF7 cells (unsync), cells treated with roscovitine (G2) or cells released from

roscovitine-block for15min (M) were analyzed by FACS with anti histone H3-

by guest on Novem

ber 19, 2020http://w

ww

.jbc.org/D

ownloaded from

27

phosphospecific antibody to quantify the M phase population. The upper-right

quadrangle represents histone H3-phosphorylated M phase.

D. Cell cycle-synchronized samples were immunoblotted with anti BRCA1 (C-20),

S308-P or actin antibodies. When released from G2 block, cell lysates were

prepared after 1h, 2h and 3h. To induce DNA damage, cells released from G2

block were immediately treated with IR (M+IR) and cell lysates were obtained

after 1h.

E. The same lysates studied in D were immunoblotted with anti-Aurora-A,

phosphorylated Aurora-A (T288-P) and actin antibodies.

FIG. 4 BRCA1 Ser308 is phosphorylated in an Aurora-A dependent manner.

A. U2OS cells were transfected with si-RNA specific for Aurora-A. After 24h, total

lysates were immunoblotted with anti Aurora-A, BRCA1 (C-20), S308-P and

actin antibodies.

B. MCF7 cells were infected with adenovirus expressing LacZ (Ad-LacZ), Aurora-A

(Ad-Aurora-A) and a kinase-defective form of Aurora-A (Ad-Aurora-A-D274N).

After 24h, cell lysates were immunoblotted with anti BRCA1, S308-P, Aurora-A

and actin antibodies.

FIG. 5 Phosphorylation of Ser308 of BRCA1 is necessary for entry into mitosis.

A. BRCA1(+/+) and BRCA1 (-/-) MEFs were infected with retrovirus expressing

LacZ, FLAG-wild type BRCA1 (WT) or FLAG-S308N BRCA1 as indicated.

by guest on Novem

ber 19, 2020http://w

ww

.jbc.org/D

ownloaded from

28

After 48h, cell lysates were immunoblotted with anti FLAG or actin antibodies to

confirm the expression of these proteins.

B. BRCA1(+/+) and BRCA1 (-/-) MEFs were infected with the indicated retrovirus

for 48h, and histone H3-phosphorylation was studied as described in the FIG. 3

caption. To induce DNA damage, after infection of MEFs with wild type (WT)

BRCA1 virus for 36h, cells were treated with IR (10Gy), and FACS analysis was

done after 12h.

by guest on Novem

ber 19, 2020http://w

ww

.jbc.org/D

ownloaded from

by guest on Novem

ber 19, 2020http://w

ww

.jbc.org/D

ownloaded from

by guest on Novem

ber 19, 2020http://w

ww

.jbc.org/D

ownloaded from

by guest on Novem

ber 19, 2020http://w

ww

.jbc.org/D

ownloaded from

by guest on Novem

ber 19, 2020http://w

ww

.jbc.org/D

ownloaded from

by guest on Novem

ber 19, 2020http://w

ww

.jbc.org/D

ownloaded from

Pat P Ongusaha, Chuxia Deng, Subrata Sen, Sam W Lee and Toru OuchiMutsuko Ouchi, Nobuko Fujiuchi, Kaori Sasai, Hiroshi Katayama, Yohji A Minamimori,

BRCA1 phosphorylation by Aurora-A in the regulation of G2 to M transition

published online February 27, 2004J. Biol. Chem. 

  10.1074/jbc.M311780200Access the most updated version of this article at doi:

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

to choose from all of JBC's e-mail alertsClick here

by guest on Novem

ber 19, 2020http://w

ww

.jbc.org/D

ownloaded from