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INTRODUCTION |
Breast cancer tumor suppressor protein BRCA1 is a nuclear
phosphoprotein with 1863 amino acids (1) that is implicated in the DNA
repair pathway and regulation of gene transcription (2-7). In normally
growing cells, BRCA1 is phosphorylated in a cell
cycle-dependent manner; the protein undergoes
hyperphosphorylation during the S phase and is dephosphorylated after
the M phase (8, 9). Biochemical analysis has revealed that Ser-1497 is
phosphorylated by cyclin-dependent kinase 2/cyclin A and E
complexes (10). It has been also shown that DNA damage induces both
nuclear redistribution of BRCA1 and an increased phosphorylation of the
protein through DNA damage-activated kinases such as
ATM,1 ATR, and hCds1/Chk2
(11-14). Several phosphorylation sites have been identified under
these conditions, including Ser-988, -1423, -1387, and -1524. It has
recently been shown that re-expression of wild type BRCA1 confers weak
resistance to DNA damage-induced cell death in HCC1937 BRCA1 mutant
breast cancer cell lines (12, 13, 15), whereas
phosphorylation-defective BRCA1 alleles carrying Ser to Ala
substitution of these residues did not rescue them from apoptosis.
Furthermore, ionizing radiation (IR) treatment has been shown to induce
phosphorylation of transcriptional corepressor CtIP, consequently
releasing the protein from the BRCA1-containing complex leading to
activation of transcriptional regulation of BRCA1 (16). These results
provide a model of how DNA damage-induced phosphorylation of BRCA1
leads to transmission of signals that regulate gene expression.
Previous studies have revealed that BRCA1 is involved in the G2-M
checkpoint (17-19). Mouse embryonic fibroblasts carrying a targeted
deletion of exon 11 of the BRCA1 gene showed a
defective G2-M checkpoint accompanying unequal chromosome segregation,
abnormal nuclear division, and aneuploidy. Interestingly, these
phenotypes were also induced by overexpression of centrosome-associated Aurora-A/BTAK/STK15 kinase, which is frequently amplified in breast cancer cells (20-22). More recently, BRCA1 has been shown to localize in the mitotic centrosome, interacting with
-tublin (23, 24). Although these studies strongly suggest pivotal functions of BRCA1 in
mitosis, physiological roles and regulation of BRCA1 phosphorylation in
mitosis still require clarification.
In the current study, we investigated how BRCA1 phosphorylation is
regulated during the cell cycle and in response to DNA damage. For
these purposes, phospho-Ser-specific antibodies recognizing Ser-988,
-1423, -1497, and -1524 residues of BRCA1 were generated and employed
to study BRCA1 phosphorylation in the S and G2/M phases of cells with
or without DNA damage. These results reinforce a model wherein
phosphorylation of specific residues of BRCA1 after DNA damage affects
its localization and function.
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EXPERIMENTAL PROCEDURES |
Generation of Phospho-specific Antibodies and Monoclonal
Antibodies for BRCA1--
Rabbit polyclonal phospho-Ser-specific
antibodies for BRCA1 were generated by Research Genetics, Inc. against
KLH-conjugated synthetic peptides as follows: S988,LFPIKSpFVKTKCK;
S1423, EPGVERSSpPSKCPS; S1497, VLEQHGSpQPSNSY; S1524,
QNRNYPSpQEELIK. Monoclonal antibody 21A8 was generated by
immunizing mice with GST-BRCA1 (amino acids 1314-1863) in the Mount
Sinai School of Medicine Core.
Western Blotting of BRCA1--
Cell extracts were prepared in
EBC buffer (50 mM HEPES, pH 7.6, 250 mM NaCl,
0.1% Nonidet P-40, 5 mM EDTA, pH 8.0) with mixed protease
inhibitor (Sigma). To detect changes in the mobility of p220-p240BRCA1,
prolonged 5% SDS-PAGE was used in 25 mM Tris, 192 mM glycine, 0.1% SDS. After transferring, filters were
blocked with 8% nonfat dried milk in TBS-T (20 mM Tris, pH
8, 0.9% NaCl, 0.5% Tween). The primary antibodies 21A8, Ab-1
(Calbiochem) and 8F7 (GeneTex) were used for 1 h at room
temperature. Antibodies S988, S1423, S1497, and S1524 were used at 10 µg/ml. The secondary antibodies (Jackson ImmunoResearch) were
peroxidase-conjugated anti-mouse IgG (H+L) or anti-rabbit IgG (H+L).
Films were developed by ECL.
Cell Culture and Preparation of Total Cell Extracts--
HEK293,
MCF7, and HCC1937 cells were obtained from ATCC. Cells were cultured in
Dulbecco's modified Eagle's medium, 10% fetal bovine serum for
HEK293 and MCF7 cells, or RPMI1640, 10% fetal bovine serum for HCC1937
cells. Cell extracts were prepared in EBC buffer (50 mM Tris, pH 8, 120 mM NaCl, 0.5% Nonidet P-40
(NP-40)), with the addition of 50 mM NaF, 1 mM
sodium orthovanadate, 100 µg/ml polymethylsulfonyl fluoride, 20 µg/ml aprotinin, and 10 µg/ml leupeptin. IR was administered using
MARK2 IRRADIATOR (J. L. Shepherd & Associates, San Fernando, CA).
Cell Fractionation--
Cells were washed with ice-cold
phosphate-buffered saline and lysed by Lysis buffer (20 mM
Hepes, pH 7.5, 20% glycerol, 10 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.1% Triton
X-100, 1 mM dithiothreitol, 1 mM
phenylmethylsulfonyl fluoride, 50 µM leupeptin, and 50 µg/ml aprotinin). After centrifuge at 2,300 rpm at 4 °C, pellet
was resuspended in nuclear extract buffer (Lysis buffer containing 500 mM NaCl). After rocking at 4 °C for 1 h, samples
were centrifuged. Twenty µg of both nuclear and cytoplasmic samples
were immunoblotted by 8F7 or nucleophosmin antibody (Cell Signaling
Technology).
Flow Cytometry--
Cell cycles were synchronized by
following a previously described protocol (26) and analyzed by
FACSCalibur (BD PharMingen).
Immunostaining Analysis--
Cells were fixed for 1 h in
phosphate-buffered saline, 3% paraformaldehyde, 2% sucrose solution,
followed by 5 min of permeabilization at room temperature in Triton
buffer (0.5% Triton X-100, 20 mM HEPES, 50 mM
NaCl, 3 mM MgCl2, 300 mM sucrose).
Blocking was done with 5% normal horse serum, 5% normal goat serum.
BRCA1 was visualized using mouse monoclonal antibody 21A8, and Ser-988
phosphorylation was visualized using the polyclonal S988 antibody. All
secondary antibodies used were species-specific antibodies from Jackson ImmunoResearch (Texas Red-X for mouse IgG and fluorescein
isothiocyanate for rabbit IgG), used at 1:100 throughout. Nuclei were
stained with DAPI. All images were collected with a Leica TCS-SP
confocal laser scanning microscope and processed using Adobe Photoshop software.
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RESULTS |
Differential Phosphorylation of BRCA1 in MCF7 Cells and
BRCA1-mutated HCC1937 Cells--
Phospho-Ser-specific antibodies were
raised and affinity purified against synthetic peptides containing
phosphorylated Ser-988 (S988), -1423 (S1423), -1497 (S1497), and -1524 (S1524) of the BRCA1 protein (see "Experimental Procedures").
Specificity of these phospho-specific BRCA1 antibodies was examined by
immunoblot analysis using cell lysates prepared from exponentially
growing MCF7 cells with or without
-phosphatase treatment (Fig.
1A, left). These
results demonstrated that S988, S1423, S1497, and S1524 antibodies
recognize only phosphorylated forms of BRCA1. Affinity-purified mouse
monoclonal antibody 21A8 was generated by immunizing mice with amino
acids 1314-1863 of the BRCA1 protein. MCF7 cells were cell cycle
synchronized, and BRCA1 phosphorylation was studied using the
antibodies described above. Increased expression of the
-form (about
240 kDa) of BRCA1 was detected in the S and G2/M phases by 21A8,
presumably as a result of the hyperphosphorylation (Fig. 1A,
right). Phosphorylation of Ser-988 and -1423 of
-form BRCA1 (about 220 kDa) weakly increased in the G2/M phase, but phosphorylation of Ser-1497 and -1524 residues did not change significantly during the cell cycle. Notably, despite high levels of
the
-form BRCA1 in the S and G2/M phases detected by 21A8, only S988
antibody weakly detected an increased signal of the
-form,
indicating that
-form BRCA1 contains multiple phosphorylation sites
other than Ser-988 in the S and G2/M phases.

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Fig. 1.
Cell cycle-dependent and DNA
damage-induced phosphorylation of BRCA1. A, specific
recognition of phosphorylated BRCA1 by S988, S1423, S1497, and S1524
antibodies. Where indicated, cell lysates were treated with 400 units
of -phosphatase (left). Phosphorylation of Ser-988,
-1423, -1497, and -1524 was studied by the indicated antibodies in
synchronized MCF7 cells (right). Positions of two major
products were shown as and . B and C,
growing cell cultures of MCF7 and HCC1937 cells were treated with UV or
IR of the indicated dosage. Cell lysates were prepared after 1 h,
and BRCA1 protein level and its phosphorylation status were studied by
the indicated antibodies.
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Next, we compared the phosphorylation of wild type and mutant forms
(5382insC) of BRCA1. Asynchronized MCF7 cells expressing wild type
BRCA1 were treated with different dosages of UV (5, 10, 50, and 100 mJ/cm2) or IR (1, 5, 10, and 50 Gy), and BRCA1
phosphorylation was then determined (Fig. 1B). Like 21A8,
anti-BRCA1 antibody 8F7 detected the
- and
-forms of BRCA1
protein in UV- and IR-treated cells. Phosphorylation of Ser-988 was
strongly induced by 1 Gy of IR or UV treatment (50-100
mJ/cm2) with slow migration at position
. UV treatment
also strongly induced Ser-1423 phosphorylation of the
-form.
Increased Ser-1524 phosphorylation was observed in the
-form of
BRCA1 in response to 10-50 mJ/cm2 of UV or 10 Gy of IR
treatment. These results suggest that DNA-damaged cells contain several
species of BRCA1 of differing phosphorylation status.
The HCC1937 breast cancer cell line expresses 5382insC mutant BRCA1,
resulting in formation of the immature BRCT domain (25). We analyzed
how this mutant form of BRCA1 is phosphorylated after DNA damage (Fig.
1C). The mutant did not show a significant mobility shift
after DNA damage. Unlike the case in MCF7 cells, both UV and IR induced
Ser-988 phosphorylation and a slight mobility shift in a
dose-dependent manner, whereas phosphorylation of Ser-1423 and -1524 was not induced to a significant degree by the same treatment. Significantly, HCC1937 cells re-expressing wild type BRCA1
have been found to be more resistant to IR damage than those expressing
S988A mutant BRCA1 (13). These results suggest that C-terminal
BRCA1 is required for phosphorylation of BRCA1 and that the functional
C-terminal BRCT domain, which is lost in mutant BRCA1 in HCC1937 cells,
plays a crucial role in survival after damage.
Phosphorylation of BRCA1 by IR in the S and G2/M Phases--
We
next investigated how DNA damage-specific phosphorylation is regulated
during the cell cycle. MCF7 cells were synchronized at the S or G2/M
phase and treated with different doses of IR as indicated. Samples were
collected 0.5, 1, 3, and 5 h after treatment, and BRCA1
phosphorylation was determined by means of S988, S1423, and S1524
antibodies. In the S phase (Fig.
2A), 21A8 consistently
detected the
- and
-forms of BRCA1. Ser-988 phosphorylation weakly increased in the
-form, but the
-form was predominantly and strongly phosphorylated in response to higher doses of treatment. Ser-1423 phosphorylation weakly increased in response to all doses tested. Ser-1524 phosphorylation was strongly induced by 5 Gy or higher
doses and sustained at least 5 h after treatment. Phosphorylation of BRCA1 in the G2/M phase was also studied (Fig. 2B).
Antibody 21A8 detected both the
- and the
-forms of BRCA1
throughout the time course. After damage, Ser-988 phosphorylation
slightly increased in the
'-form detected between the
- and
-forms. Ser-1423 was phosphorylated in the
-form as early as
3 h by 1 Gy of radiation, but 5 Gy or higher doses induced Ser-423
phosphorylation within 30 min. Ser-1524 phosphorylation did not
increase significantly in the
-form, and 10 Gy or higher doses of
radiation even decreased phosphorylation.

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Fig. 2.
IR-induced BRCA1 phosphorylation in S and
G2/M phase. MCF7 cells were cell cycle synchronized at S
(A) or G2/M (B) phase and treated with different
dosages of IR. Samples were prepared after 0.5, 1, 3, and 5 h, and
the phosphorylation status was studied by the indicated antibodies. The
molecular weight of the - and -forms corresponds to those in Fig
1.
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These results demonstrate that (i) phosphorylation sites of BRCA1 are
determined by both the level of DNA damage and the time course of
unfolding of the effects of that damage, and (ii) after DNA damage
BRCA1 exists as a heterogeneous pool of species with differing
phosphorylation status even when the cell cycle has been synchronized.
Phosphorylation of BRCA1 by UV Radiation in the S and G2/M
Phases--
We next studied UV-induced BRCA1 phosphorylation.
Monoclonal antibody 21A8 predominantly detected the
-form of BRCA1
in damaged cells in the S phase (Fig.
3A). Reduced expression of
BRCA1 in cells treated with high UV doses is presumably because of
protein degradation of apoptotic cells. The BRCA1
"-form, similar in molecular weight to the
'-form in Fig. 2B, showed Ser-988
phosphorylation when treated with 50 and 100 mJ/cm2 of UV
after 3-5 h. Ser-1423 phosphorylation did not change in response to
the lower dose but slightly decreased with 50 and 100 mJ/cm2 of UV. No significant changes of Ser-1524
phosphorylation were detected.

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Fig. 3.
UV-induced BRCA1 phosphorylation in S and
G2/M phase. MCF7 cells were cell cycle synchronized at S
(A) or G2/M (B) phase and treated with different
dosages of UV. Immunoblot analysis was done by the same procedures
described in Fig 2. The "-form represents the molecular
weight between - and -forms shown in Fig 1.
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Antibody 21A8 was found to recognize hyperphosphorylated BRCA1
(
-form) in the G2/M phase, although the antibody had been generated
by immunizing a bacterial GST fusion protein that presumably is an
unphosphorylated peptide (Fig. 3B). S988 and S1423 detected doublet signals (
- and
-forms) when treated with 50 and 100 mJ/cm2 of UV. In the
-form Ser-1524 phosphorylation was
slightly increased by 50 mJ/cm2 of UV. Again, these results
demonstrate that several species of BRCA1 with differential
phosphorylation status are present in UV-damaged S and G2/M phase cells.
Mitosis-specific Localization of Ser-988-phosphorylated
BRCA1--
Cell cycle-dependent localization of BRCA1 was
determined in detail by confocal laser microscopy using the 21A8 and
S988 antibodies (Fig. 4A). As
reported previously, nuclear BRCA1 was weakly stained during the G0 and
G1 phases, and a distinct dot pattern appeared during S phase (26). A
large nuclear dot pattern was induced 1 h after cells were treated
with IR or UV but disappeared after 5 h. Although weak cytoplasmic
dots were recognized by S988 during S phase, UV or IR damage
induced a perinuclear dot pattern and nuclear signal. Redistribution of
BRCA1 in DNA-damaged cells was confirmed by subcellular fractionation
(Fig. 4B). Nuclear and cytoplasmic extracts of S phase cells
were prepared at 1 h post-UV and -IR treatment. Although BRCA1 was
predominantly nuclear in untreated S phase cells, DNA damage induced
cytoplasmic redistribution of BRCA1.

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Fig. 4.
Cell cycle-dependent and DNA
damage-induced localization of Ser-988-phosphorylated BRCA1 in MCF7
cells. A, confocal microscopy was employed to determine
the distribution of BRCA1 protein under conditions of DNA damage.
S phase cells were treated by UV (10 mJ/cm2) or IR
(5 Gy) and immunostained after 1 or 5 h. Arrows
indicate the centrosome in metaphase and the cleavage furrow in
cytokinesis, respectively. B, subcellular fractionation of
BRCA1 in S phase cells with or without DNA damage (UV, 10 mJ/cm2; IR, 5 Gy). Samples were prepared 1 h after
damage and immunoblotted by antibodies for BRCA1 (8F7) or
nucleophosmin.
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Although BRCA1 was visualized in both chromosome and cytoplasm during
mitosis, S988 detected BRCA1 localized to the centrosome and
microtubules only during metaphase; BRCA1 localization to the inner
structure of the chromosome was also detected during anaphase and
telophase. Interestingly, Ser-988-phosphorylated BRCA1 was also
detected in the cleavage furrow during telophase and cytokinesis. These
results suggest that specific phosphorylation of BRCA1 may be important
for the regulation of mitosis.
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DISCUSSION |
In the present studies, generation of S988, S1423, S1497, and
S1524 antibodies enabled us to further investigate the regulation of
BRCA1 phosphorylation in different conditions. Our results demonstrate
that BRCA1 exists as a heterogeneous species of differentially phosphorylated status and that phosphorylation of each Ser residue examined is specifically induced in cell cycle and in response to DNA
damage. Thus, at damaged state, BRCA1 exists as a pool of species with
different phosphorylation status. It is likely that phosphorylation of
specific residues determines a role and localization of BRCA1 in
damaged cells.
Human Cds1 kinase (hCds1/Chk2) has been shown to colocalize BRCA1 and
phosphorylate Ser-988 residue (12). In that study, low dosage of
-radiation (10 Gy) was sufficient to induce Ser-988 phosphorylation.
Our results were consistent with these data in that Ser-988
phosphorylation was detected by 1 Gy of ionizing radiation in MCF7
cells. We also found that phosphorylation of BRCA1 Ser-988 is increased
when IR-damaged in S phase (Fig. 2A), whereas it is not
obvious in G2/M phase (Fig. 2B) although Chk2 kinase can
respond to DNA damage throughout the cell cycle (27). It remains
unclear whether hCds1/Chk2 colocalizes BRCA1 in G2/M phase.
Significantly, HCC1937 cells re-expressing wild type BRCA1 have been
found to be more resistant after
-radiation damage than those
expressing S988A mutant BRCA1 (12). Our studies show that Ser-988 of
truncated BRCA1 in HCC1937 cells is similarly phosphorylated with wild
type BRCA1 in MCF7 cells. Taken together, these studies provide an
implication that the phosphorylation of Ser-988 is not sufficient for
damage-resistant phenotype and that the functional C-terminal BRCT
domain, which is lost in mutant BRCA1 in HCC1937 cells, plays a crucial
role for survival after damage.
Several other Ser residues have been identified as potential
phosphorylation sites by ATM, including Ser-1387, -1423, -1457, and
-1524 (13, 28, 29). Ser-1423 and -1524 are associated with the
regulation of cell growth after IR; HCC1937 cells re-expressing wild
type BRCA1 can grow after IR damage, whereas cells expressing a
phosphorylation-deficient mutant of these Ser residues show growth
retardation under the same condition (13, 15). It is tempting to
speculate that the allosteric change of BRCA1 structure because of
phosphorylation affects the interaction between BRCA1 and other
proteins involved in the DNA damage response. Among them are BRCA2 and
the Rad50-Mre11-Nbs complex, but it has been found that
damage-dependent phosphorylation does not change the amount
of BRCA1 associated with these complexes (30, 31).
We found that DNA damage induces both nuclear dot pattern and
perinuclear distribution of Ser-988-phosphorylated BRCA1. Recently, it
was reported that BRCA1 can shuttle between nucleus and cytoplasm and
that a Rev-type nuclear export sequence near the N terminus facilitates
export through the CRM exporting pathway (32). These results
have also shown that DNA damage (actinomycin D treatment) resulted in
the accumulation of BRCA1 in the cytoplasm, suggesting that DNA damage
induces shuttling of the BRCA1 protein, although whether Ser-988
phosphorylation is necessary for the relocalization of the BRCA1
protein remains to be determined. Studies are in progress to elucidate
whether Ser-988 can be phosphorylated when export is inhibited by
Leptomycin B, a specific inhibitor of CRM-dependent BRCA1
nuclear export1.
Antibody 21A8 detected chromosome structure throughout mitosis, and
Ser-988-phosphorylated BRCA1 was detected in both cytoplasm and the
inner mitotic chromosome in prophase, anaphase, and telophase. In
metaphase, centrosome and spindle structures were strongly stained by
S988. Although we could not determine precise localization of
Ser-988-phosphorylated BRCA1 in the inner chromosome, this localization
may represent the mitosis-specific structure, such as the kinetochore.
Supporting this hypothesis, BRCA2, which has been shown to colocalize
with BRCA1 in mitosis (29), was shown to associate with hBUBR1, a
component of the kinetochore CEMP-E/F complex (33).
In summary, the present results demonstrate that BRCA1 phosphorylation
sites in response to DNA damage are determined by the cell cycle and
the dosage of the damage and that BRCA1 exist as a heterogeneous pool
of species with differential phosphorylation status even when cell
cycle has been synchronized. Our results raised several issues that are
potentially important for the tumor suppressive function of BRCA1 in
the DNA damage pathway; biochemical details of signaling conferred by
phosphorylated BRCA1 will be necessary to understand the role of this
protein in this pathway.