From the Molecular and Cell Biology Laboratory and
¶ Laboratory of Genetics, The Salk Institute for Biological
Studies, La Jolla, California 92037 and the
Department of
Biological Chemistry, UCD Cancer Center/Basic Science, University
of California at Davis, Sacramento, California 95817
Received for publication, May 10, 2002, and in revised form, November 12, 2002
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ABSTRACT |
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The breast and ovarian cancer-specific tumor
suppressor RING finger protein BRCA1 has been identified as an E3
ubiquitin (Ub) ligase through in vitro studies, which
demonstrated that its RING finger domain can autoubiquitylate and
monoubiquitylate histone H2A when supplied with Ub, E1, and UBC4 (E2).
Here we report that the E3 ligase activity of the N-terminal 110 amino
acid residues of BRCA1, which encodes a stable domain containing
the RING finger, as well as that of the full-length BRCA1, was
significantly enhanced by the BARD1 protein (residues 8-142), whose
RING finger domain itself lacked Ub ligase activity in
vitro. The results of mutagenesis studies indicate that the
enhancement of BRCA1 E3 ligase activity by BARD1 depends on direct
interaction between the two proteins. Using K48A and K63A Ub mutants,
we found that BARD1 stimulated the formation of both Lys48-
and Lys63-linked poly-Ub chains. However, the enhancement
of BRCA1 autoubiquitylation by BARD1 mostly resulted in poly-Ub chains
linked through Lys63, which could potentially activate
biological pathways other than BRCA1 degradation. We also found that
co-expression of BRCA1 and BARD1 in living cells increased the
abundance and stability of both proteins and that this depended on
their ability to heterodimerize.
BRCA1 is a tumor suppressor gene that is mutated
in 50-90% of hereditary breast and ovarian cancers (1, 2). The human BRCA1 gene encodes a large protein of 1863 amino acids,
which contains an N-terminal RING-finger domain and two C-terminal BRCT domains (3, 4). To date, BRCA1 has been implicated in interactions with
more than 20 proteins and involved in a remarkable range of cellular
processes from transcriptional regulation to DNA damage repair (5-10).
It has been suggested that BRCA1 may regulate various biological
pathways via a common mechanism such as chromatin remodeling (11-14).
20% of the clinically relevant mutations of BRCA1 occur within the
N-terminal 100 residues, which contain the RING motif (residues 23-76)
(15). Recently, RING domains have been documented to have E3 ubiquitin
ligase activity (16, 17). The RING E3s function as adaptors to recruit
substrates and a Ub-conjugating enzyme (E2),1 and to mediate the
transfer of Ub from E2 to substrate proteins (16-18). Ub conjugation
(ubiquitylation) is well known as a signal for protein degradation and
is involved in multiple biological pathways (19). The RING domain of
BRCA1 exhibits E3 ligase activity in vitro (20-22), and all
of the cancer predisposing mutations in the RING domain that have been
tested inactivate BRCA1 E3 Ub ligase activity (21, 22).
Human BARD1 (BRCA1-associated
RING domain 1) encodes a protein of
777 amino acids and contains an N-terminal RING domain (residues 49-100) and two C-terminal BRCT motifs (15, 23). In living cells,
BRCA1 exists mostly as a heterodimeric complex with BARD1 (23, 24). The
recently reported NMR structure of a BRCA1-BARD1 heterodimeric complex
reveals that the Here we report that BARD1 can significantly enhance BRCA1 E3 Ub ligase
activity by directly binding BRCA1, although BARD1 itself does not
exhibit E3 ligase activity in vitro. BARD1 and BRCA1 form
heterodimers in living cells and mutually control each other's
abundance and stability. The enhancement of BRCA1 autoubiquitylation by
BARD1 mostly results in poly-Ub chains linked through
Lys63, which could be involved in pathways related to DNA
damage response and repair rather than signaling BRCA1 degradation.
Plasmid Constructs--
A DNA fragment containing the N-terminal
110 amino acids of BRCA1 was PCR-amplified from pBluescript II SK(+)
73.1 (35) using the primers (5'-GCCGGATCCATGGATTTATCTGCTCTTCGC-3') and
(5'-GGCGAATTCCTTACTTTTTTGCAAAATTATAGC-3'). The fragment was cloned
using BamHI and EcoRI into pGEX-KG and pHis8 to
create pYN122 and pYN131, respectively. A DNA fragment containing the
N-terminal 308 amino acids of BRCA1 was PCR-amplified from pBluescript
II SK(+) 73.1 using the primers
(5'-GCCCACTAGTATGGATTTATCTGCTCTTCGC-3') and
(5'-GAAATGCGGCCGCTCAGAATTCAGCCTTTTCTACAT-3') and cloned using SpeI and NotI into pFLAG to generate pYN146.
Based on the plasmid B230AE/pGEX, which contains WT GST-tagged
N-terminal 8-142 aa of BARD1 (a gift from Dr. Richard Baer), plasmids
pYN124 (C83G), pYN128 (C50G), pYN129 (R58A), pYN130 (I69A), pYN132
(H68A), pYN141 (H68A/C83G), pYN142 (C50G/H68A), pYN143
(C50G/C83G), pYN156 (L44R), and pYN158 (I105D) were created using the
QuikChange site-directed mutagenesis kit (Stratagene). A DNA
fragment containing the N-terminal 8-142 aa of BARD1 was PCR-amplified
from B230AE/pGEX using primers (5'-GCCCACTAGTCCTCGAGGCCACGAAG-3') and
(5'-GAAATGCGGCCGCTCACGATGAATTCTTCTTG-3') and cloned using
SpeI and NotI into pFLAG to generate pYN125. DNA
fragment containing full-length BARD1 was PCR-amplified from BARD1-m1/pSP6 using primers
(5'-GAAATGCGGCCGCTCAGCTGTCAAGAGGAAGC-3') and
(5'-GCCCACTAGTATGCCGGATAATCGGC-3') and cloned using
SpeI and NotI into pFLAG to generate pYN147.
Based on pYN147, pYN148 (C50G), pYN149 (R58A), pYN150 (H68A), pYN151
(I69A), pYN152 (C83G), pYN153 (C50G/C83G), pYN154 (C50G/H68A),
pYN155 (H68A/C83G), pYN159 (L44R), and pYN161 (I105D) were generated
using the QuikChange site-directed mutagenesis kit. The plasmid
expressing GST-BRCA1-(1-78) has been described previously (21). The
plasmid pHIV CSC was constructed by introducing the cytomegalovirus
promoter from pEGFP-C1 (Invitrogen) into the pHIV CS vector (36).
Subsequently, the full-length BRCA1 cDNA was introduced into the
pHIV CSC vector from pBluescript 73.1, giving rise to pHIV CSC BRCA1.
The baculovirus plasmid expressing full-length BRCA1 was generated by
inserting BRCA1 cDNA into pAcSG2 (Pharmingen) with FLAG
sequence tagged at the N terminus of BRCA1.
Expression and Purification of Proteins and GST Pull-down
Assays--
GST-tagged and His-tagged proteins were expressed and
purified as described by Leverson et al. (37). GST-tagged
proteins were expressed in Escherichia coli strain BL21
(DE3) by induction with 0.4 mM
isopropyl-1-thio-
His-tagged proteins were expressed as described above and purified by
using Talon metal affinity resin (Clontech). Cells
were lysed in binding buffer (20 mM Tris-Cl, pH 7.5, 100 mM NaCl, 10% glycerol, 10 µM
ZnSO4, and 1 mM imidazole) and bound to a 1-ml bed volume of Talon resin. After washing with 10 bed volumes of binding buffer plus 10 mM imidazole, the bound proteins
were eluted with binding buffer plus 100 mM imidazole.
Baculovirus FLAG-tagged full-length BRCA1 was generated and purified
essentially as described (39). The concentration of purified BRCA1 was
~0.1 mg/ml and judged to be 95% pure as evidenced by a single
Coomassie-stained band after resolution by Tris acetate 3-8% SDS-PAGE
(Novex/Invitrogen).
In Vitro Ubiquitylation Reactions--
In vitro
ubiquitylation assays were carried out as previously described (40),
using purified bacterially expressed His-E1 and His-Ubc4. About 1 µg
of purified GST-BRCA1-(1-110) was incubated with 50-500
nM His-E1, 0.5-5 µM His-Ubc4, 10 µM bovine ubiquitin or GST-Ub (WT or mutant), and 2 mM ATP in reaction buffer (50 mM Tris-Cl, pH
7.5, 2.5 mM MgCl2, and 0.5 mM
dithiothreitol). Purified GST-BARD1-(8-142) (WT or mutant) was added
to the reactions as indicated. After a 90-min incubation at room
temperature, reactions were stopped with 2 × SDS buffer,
separated by SDS-PAGE, and analyzed by immunoblotting with anti-Ub
monoclonal antibody (Zymed Laboratories Inc.),
anti-GST monoclonal antibody (Santa Cruz Biotechnology, Inc., Santa
Cruz, CA), anti-His5 monoclonal antibody (Qiagen), or
affinity-purified anti-BRCA1 (A) polyclonal antibodies (41).
Immunoblots to detect histone ubiquitylation were performed with a
monoclonal anti-Ub antibody (Zymed Laboratories Inc.)
and rabbit polyclonal anti-H2A, anti-H2B, anti-H3, and anti-H4
antibodies (Upstate Biotechnology). 3 µg of Drosophila
core histones (a gift from Joaquin Espinosa and Beverly Emerson,
Regulatory Biology Laboratory, The Salk Institute for Biological
Studies) and 0.25 µg of full-length FLAG-BRCA1 were added to
each reaction in the absence or presence of 0.5 µg of
GST-BARD1-(8-142).
Cell Culture and DNA Transfection--
293T human embryonic
kidney cells were maintained in Dulbecco's modified Eagle's medium
supplemented with 10% fetal bovine serum and antibiotics. DNA
transfections were carried out by a standard calcium phosphate
precipitation protocol. In all of the transfections, the total amount
of DNA was equalized. 48 h post-transfection, the cells were
collected and lysed in radioimmune precipitation buffer (6 mM Na2HPO4, 4 mM
NaH2PO4, 2 mM EDTA, 150 mM NaCl, 1% Nonidet P-40, 1% sodium deoxycholate, 0.1%
SDS, 1% Trasylol, 50 mM NaF, and 100 µM
Na3VO4), and equal amounts of proteins were analyzed by SDS-PAGE. To examine the relative stability of BRCA1 and
BARD1 proteins, fresh media containing cycloheximide (150 µg/ml final
concentration) were added 48 h after transfection. The cells were
collected at the times indicated, and cell lysates were subjected to
ECL immunoblot analysis. The membranes were probed with anti-FLAG
(Sigma) and anti-GST-Nck-
Transfection of full-length BRCA1 into 293T cells was carried out by
the calcium phosphate BBS transfection method (42). After lysis, the
samples were separated using Novex/Invitrogen 3-8% Tris-acetate
SDS-PAGE gels and blotted according to the manufacturer's recommendation except for transfer times, which were extended to 90 min. Primary antibodies were diluted in 3% bovine serum albumin/PBST
at a 1:1000 dilution for the anti-BRCA1 MS110 (Ab-1; Oncogene Science)
and 1:5000 for anti- The E3 Ubiquitin Ligase Activity of BRCA1 Is Enhanced in the
Presence of BARD1--
The BRCA1 central RING motif, which encompasses
residues 23-76 (15), is part of a larger proteolysis-resistant
structural domain containing the first 110 residues of BRCA1 (43). A
plasmid expressing GST-BRCA1-(1-110) was constructed, and bacterially expressed protein was purified using glutathione beads. Purified GST-BRCA1-(1-110) was assayed for its ability to mediate the transfer of Ub and stimulate the synthesis of stable Ub conjugates in an in vitro ubiquitylation assay, using blotting with an
anti-Ub monoclonal antibody to detect ubiquitylated products. As shown in Fig. 1B, the BRCA1 RING
domain exhibited E3 Ub ligase activity in an E1- and
E2-dependent manner, consistent with previously published
results (21). The polyubiquitylated conjugates include ubiquitylated
BRCA1 and His-E1/E2 (data not shown). Purified His-BRCA1-(1-110) had
E3 ligase activity similar to that obtained with GST-BRCA1-(1-110) (data not shown). Histone H2A could also be monoubiquitylated by
GST-BRCA1-(1-110) (data not shown) and by full-length BRCA1 (Fig.
1C, lane 7). The band was identified
as monoubiquitylated H2A, based on its reactivity with both anti-H2A
and anti-Ub antibodies. When the same membrane was probed with
anti-H2B, anti-H3, and anti-H4 antibodies, no extra slower migrating
bands were observed (data not shown), indicating that H2A is
specifically monoubiquitylated by BRCA1 in vitro.
To examine whether BARD1, which is itself a RING protein, might affect
BRCA1 E3 Ub ligase activity, increasing amounts of purified
GST-BARD1-(8-142) containing the RING domain (residues 49-100) were
incubated together with GST-BRCA1-(1-110), His-E1, His-E2 (Ubc4), and
GST-Ub. BARD1 significantly enhanced GST-BRCA1 E3 Ub ligase activity
(Fig. 1B), as had previously been reported in analogous
studies by Hashizume et al. (22) and Chen et al. (33). His-BRCA1-(1-110) E3 ligase activity was also greatly stimulated
by BARD1 (data not shown). The GST-BARD1-(8-142) protein lacked E3
ligase activity in vitro, even when present in the assay at
much greater levels than BRCA1 (Fig. 1D, lanes
6-10). As shown in Fig. 1C, the
monoubiquitylation of histone H2A by full-length BRCA1 was also much
stronger in the presence of BARD1 (lanes 7 and
9), consistent with the result from Pan's group
(33). In the presence of BARD1, several slowly migrating bands
appeared when the membrane was probed with anti-Ub antibody, which
could be polyubiquitylated products. However, when the same membrane was probed with anti-H2A antibodies, only one weak extra band migrating
slower than monoubiquitylated H2A was observed (Fig. 1C,
lane 9). Therefore, we conclude that histone H2A
was predominantly monoubiquitylated by BRCA1 in vitro in the
presence of BARD1.
The enhancement of BRCA1 E3 Ligase Activity by BARD1 Depends on
Direct Interaction between the Two Proteins--
To determine whether
the enhancement of BRCA1 E3 Ub ligase activity by BARD1 depends on the
integrity of the BARD1 RING domain and the interaction between the two
proteins, the RING consensus residues Cys50,
Cys83, and His68, in the BARD1 RING domain were
mutated to Gly or Ala; the nonconserved Arg58 and
Ile69 were also mutated to Ala (Fig.
2A). The conserved Cys and His residues are necessary for the integrity of the BARD1 RING domain, which is in turn required for the proper orientation of the N- and
C-terminal helices that form the four-helix bundle, and therefore these
mutations might be expected to affect the interaction between BRCA1 and
BARD1. The mutant GST-BARD1-(8-142) proteins were then purified and
examined for their ability to bind BRCA1. GST protein and WT
GST-BARD1-(8-142) were used as negative and positive controls, respectively. As shown in Fig. 2B, WT GST-BARD1-(8-142)
bound strongly to His-BRCA1-(1-110), whereas GST itself did not. The R58A and I69A mutant proteins bound equally well to BRCA1 when compared
with WT BARD1. The C50G, H68A, and C83G single mutant proteins still
bound to BRCA1 weakly, whereas the C50G/H68A, C50G/C83G, and H68A/C83G
double mutant proteins only exhibited extremely low binding activity in
the GST pull-down assay (Fig. 2B). To test the importance of
the hydrophobic interactions in the four-helix bundle that stabilize
the BRCA1-BARD1 heterodimer, we also constructed two BARD1 mutants with
mutations in critical hydrophobic residues in the N- or C-terminal
Next, equal amounts of purified WT or mutant BARD1 proteins were tested
for their effects on BRCA1 E3 Ub ligase activity using GST-Ub, and
ubiquitylated products were detected by blotting with anti-GST
monoclonal antibody. As shown in Fig. 3,
WT GST-BARD1-(8-142) and the R58A and I69A mutant proteins
significantly enhanced BRCA1 E3 Ub ligase activity (lanes
4, 6, and 8). The C50G, H68A, and C83G
mutant proteins also enhanced BRCA1 E3 ligase activity, although relatively weakly (lanes 5, 7, and
9). The I105D, C50G/H68A, and C50G/C83G mutant proteins had
only very small stimulatory effects (lanes
12-14), and the L44R mutant protein had almost no
detectable effect on BRCA1 E3 Ub ligase activity (Fig. 3,
lane 11). In addition, the same amount of
purified GST-BARD1-(8-142) had only an extremely weak stimulatory
effect on the E3 ligase activity of GST-BRCA1-(1-78) (Fig. 3,
lanes 15 and 16), which lacks the
C-terminal BRCA1 and BARD1 Mutually Stabilize Each Other in Vivo--
To
further investigate the molecular basis of BRCA1 and BARD1 cooperation,
expression plasmids for FLAG-tagged human BRCA1 (full-length or
N-terminal 308 amino acids) and FLAG-tagged WT or mutant human BARD1
(full-length or aa 8-142) were co-transfected into human 293T cells.
As shown in Fig. 4A, the
levels of FLAG-BRCA1-(1-308) protein were dramatically increased in
the presence of increasing amounts of FLAG-tagged WT BARD1 (full-length
or aa 8-142). To test the effect of BARD1 on full-length BRCA1, pHIV
CSC BRCA1, which expresses full-length human BRCA1 in the third
generation lentiviral vector, was co-transfected with FLAG-tagged
BARD1 (full-length) expression plasmid or pBluescript II (KS+)
(Stratagene) control plasmid. pCMX LacZ (45) and pEGFP C-2
(Clontech) were also co-transfected as controls for
transfection efficiency. The level of transfected full-length BRCA1
protein was significantly elevated when full-length FLAG-BARD1 was
co-expressed (Fig. 4B). The endogenous BRCA1 protein level
was also increased by transfected BARD1 (data not shown).
Next, the effects of mutant BARD1 proteins on BRCA1 abundance were
assessed. As shown in Fig. 4C, the levels of
FLAG-BRCA1-(1-308) protein were dramatically increased when
full-length WT BARD1 or the R58A mutant was co-expressed
(lanes 2, 4, and 13). In
contrast, the C50G, H68A, and C83G single mutant proteins caused a
relatively small increase in BRCA1 levels (lanes
3, 5, and 6), whereas the C50G/H68A,
C50G/C83G, and H68A/C83G double mutant and L44R and I105D mutant
proteins only had an extremely small stimulatory effect on BRCA1
abundance (Fig. 4C, lanes 7-9,
11, and 12). Mutant BARD1-(8-142) proteins
behaved similarly to mutant BARD1 (full-length) (data not shown). The
effect of BRCA1 on BARD1 abundance was also assayed. As shown in Fig.
4D, the levels of WT FLAG-BARD1 (full-length or aa 8-142)
proteins were significantly elevated in the presence of increasing
amounts FLAG-BRCA1-(1-308).
To investigate whether the increased abundance of co-expressed BRCA1
and BARD1 was due to their ability to stabilize each other in
vivo, the cells were treated with the protein synthesis inhibitor
cycloheximide 48 h after transfection. When WT FLAG-BARD1 (full-length) was co-expressed with WT FLAG-BRCA1-(1-308), both proteins were almost completely stable for up to 6 h (Fig.
5, lanes 11-15).
When the BARD1 C50G/H68A double mutant was co-expressed with
FLAG-BRCA1, the levels of both proteins were decreased by 30 min, and
they were essentially completely degraded by 3-6 h (Fig. 5,
lanes 6-10). Full-length FLAG-BARD1 (C50G/H68A)
seemed more unstable than FLAG-BRCA1-(1-308). The disruptive effect of the double mutation in the BARD1 RING domain (C50G/H68A) may contribute to its more rapid degradation. Similar to the RING finger mutants, FLAG-BARD1 (L44R) was not stabilized when it was co-expressed with
FLAG-BRCA1-(1-308) (data not shown). When BARD1 or BRCA1 were
co-transfected with vector control pFLAG, the levels of both proteins
were very low, and they were rapidly degraded (Fig. 5, lanes
1-5 and 16-20). The results suggest that BRCA1
and BARD1 stabilize each other through their direct interaction.
BARD1 Enhances BRCA1 Autoubiquitylation Involving Polyubiquitin
Chains Linked through Lys63--
It would be paradoxical
if the enhancement of BRCA1 autoubiquitylation by BARD1 led to the
degradation of BRCA1, since our data indicated that these proteins
actually stabilize each other. Poly-Ub chains can be linked through Lys
residues 11, 29, 48, and 63 in vivo (46).
Lys48-linked chains target proteins to the proteasome,
whereas Lys63-linked chains function to signal biological
processes other than proteasome-mediated degradation (46-48). To
investigate whether BRCA1 E3 ligase activity promotes formation of
Lys48- or Lys63-linked poly-Ub chains and
whether this is influenced by BARD1, K48A, K63A, and K48A/K63A GST-Ub
mutants were generated. BRCA1 E3 Ub ligase activity was assayed in the
absence or presence of GST-BARD1-(8-142) using equal amounts of
purified GST-tagged wild type Ub, Ub(K48A), Ub(K63A), or Ub(K48A/K63A)
in each reaction. The levels of poly-Ub chains were assayed by blotting
with anti-BRCA1 (A) antibodies and then reprobing with anti-Ub
antibody after stripping. Double mutant GST-Ub(K48A/K63A) was almost
inactive in the in vitro ubiquitylation assay (Fig.
6, lanes 9 and
10), indicating that most of the poly-Ub chains were linked
through Lys48 or Lys63. With GST-Ub and
GST-Ub(K63A), the signals for poly-Ub conjugates were much stronger in
the presence of GST-BARD1-(8-142), when the membrane was probed with
anti-Ub antibody (Fig. 6, left panel, lanes 3, 4, 7, and
8). For GST-Ub(K48A), the signals for poly-Ub proteins were
also stronger with GST-BARD1-(8-142) although much weaker compared
with those for GST-Ub and GST-Ub(K63A) (Fig. 6, left
panel, lanes 5 and 6). This
indicated that BARD1 dramatically enhanced BRCA1 E3 Ub ligase activity
and that formation of both Lys48 and Lys63
poly-Ub chains was stimulated by BARD1, although most of the poly-Ub
chains detected in the reaction were linked through Lys48
(Fig. 6, left panel).
A different picture emerged when the membrane was probed with
anti-BRCA1 (A) antibodies. In the absence of BARD1, signals for
BRCA1-(GST-Ub)n conjugates were much stronger with GST-Ub (WT)
and GST-Ub (K63A) than with GST-Ub (K48A), indicating that most of the
poly-Ub chains attached to BRCA1 were linked through Lys48
in the absence of BARD1 (Fig. 6, right panel,
lanes 3, 5, and 7).
However, the signals obtained with GST-Ub(K48A) and GST-Ub(K63A) in the
presence of GST-BARD1-(8-142) were nearly equivalent, and the signal
observed with GST-Ub(K63A) was only weakly stimulated by
GST-BARD1-(8-142), whereas that observed with GST-Ub(K48A) was
strongly stimulated by GST-BARD1-(8-142) (Fig. 6, right
panel, lanes 5-8). This suggests that
BARD1 stimulation of BRCA1 autoubiquitylation results in
Lys63-linked poly-Ub chains, although a significant
fraction of total BRCA1 autoubiquitylation occurs through
Lys48 linkages. BRCA1-stimulated ubiquitylation of other
proteins in the reaction results in formation of both
Lys48- and Lys63-linked poly-Ub chains, but
this occurs primarily on Lys48. Probing the membrane with
anti-His5 monoclonal antibody showed that the poly-Ub
conjugates included ubiquitylated His-E1/E2 in addition to
ubiquitylated BRCA1 (data not shown), which in part explains the
different patterns obtained when the membrane was probed with anti-Ub
and anti-BRCA1 (A) antibodies (Fig. 6). It is also to be expected that
anti-Ub antibody will detect polyubiquitylated protein/BRCA1 more
efficiently than anti-BRCA1 (A), because each polyubiquitylated BRCA1
molecule has multiple sites for binding Ub antibody but only one site
for binding BRCA1 antibodies (the BRCA1 (A) antibodies used in the
assay was directed against BRCA1 amino acids 2-20). To rule out the
possibility that GST itself is the target for poly-Ub chain attachment,
the GST-tagged wild type and mutant Ub were incubated with thrombin to
cleave off the GST tag. WT and mutant Ub proteins without GST tags were
then used in the in vitro ubiquitylation assay. The results
were similar to those obtained with GST-tagged Ub (data not shown).
This result, plus the fact that GST-BARD1 cannot be polyubiquitylated
in vitro by BRCA1 (data not shown), indicated that GST is
not a substrate for BRCA1 E3 Ub ligase.
The RING finger domain is a zinc-binding motif, characterized by a
set of spatially conserved Cys and His residues that follow the linear
order C3HC/HC3 within the primary amino acid sequence (49). RING
domains can specifically interact with E2 Ub-conjugating enzymes,
thereby promoting ubiquitylation (16, 17). Previously, the BRCA1 RING
finger was demonstrated to have in vitro E3 ligase activity
that depended on an intact RING finger structure (20-22), and the E3
ligase activity of the BRCA1 RING finger was shown to be greatly
enhanced by the BARD1 RING finger domain by Ohta's and Pan's groups
(22, 33), presumably dependent on the stable complex formed between
these two domains (15, 50). In the present study, we showed that the
BARD1 RING domain protein enhances BRCA1 RING domain E3 Ub ligase
activity through direct interaction between the two proteins. Moreover,
we found that the BARD1 enhancement of BRCA1 autoubiquitylation mostly
resulted in the formation of Lys63-linked Ub chains, whose
significance is discussed below. Consistent with previous results,
GST-BRCA1-(1-110), which encodes a stable domain containing the RING
motif, exhibited E3 Ub ligase activity in vitro, which
mediated autoubiquitylation or monoubiquitylation of histone H2A
(Fig. 1, B and C). However, the RING domain of BARD1 protein lacked E3 ligase activity in vitro, even when
used at much higher concentrations than BRCA1 (Fig. 1D). The
NMR structure of BARD1 in association with BRCA1, recently reported by
Klevit's group (15), reveals that BARD1 lacks the central Analysis of BARD1 RING finger mutants indicated that the conserved Cys
and His residues in the RING domain are important for BARD1 binding to
BRCA1 (Fig. 2B), probably because an intact RING domain is
needed to orient the N- and C-terminal helices involved in
heterodimerization, and that the enhancement of BRCA1 E3 Ub ligase
activity by BARD1 is completely dependent on direct interaction between
the two proteins (Fig. 3). This conclusion is supported by the
deleterious effects of mutating L44R and I105D, which are part of the
hydrophobic core forming the BARD1:BRCA1 interface and one of the
intramolecular helical packing contacts, respectively, and whose
mutation abolishes BARD1 binding to BRCA1 (15, 44). Moreover, the
direct interaction between BARD1 and BRCA1 appears to be more important
than the integrity of the BARD1 RING domain for enhancement of BRCA1 E3
ligase activity (Fig. 3). The enhancement of BRCA1 E3 ligase activity
by BARD1 could result from a conformational change in the BRCA1 RING
finger induced upon BARD1 binding that promotes a functional
interaction with the E2 Ub-conjugating enzyme.
In transient expression studies, we found that BRCA1 levels were
dramatically elevated by co-expressed BARD1 (Fig. 4, A and B) and that the stimulatory effect was dependent on the
interaction between the two proteins (Fig. 4C). Likewise,
BARD1 levels were elevated by co-expressed BRCA1 (Fig. 4D),
which is consistent with the report from Ohta's group (22). The
increased abundance of both proteins was due to increased stability,
and this effect required direct interaction between BRCA1 and BARD1
(Fig. 5). This may explain why BRCA1 exists in vivo in the
form of a BRCA1/BARD1 heterodimer (23, 24). Our results are in
agreement with the report from Livingston's group, in which they
showed that Xenopus laevis BRCA1 and BARD1
mutually stabilize each other through their interaction (52). The
stabilization of both proteins seems likely to be due to the highly
stable conformation of the heterodimer. Stabilization of one RING
domain protein by another has been observed previously; the MDM2 E3 Ub
ligase and the structurally related protein MDMX interact through their
RING finger domains, and this interaction stabilizes MDM2 and prevents
it from degradation (53, 54). These examples suggest an interesting
possibility that RING-RING interactions might be a general regulatory
feature of RING finger E3 Ub ligases.
The fact that BARD1 binds BRCA1 and stabilizes it in vivo
and yet enhances its E3 Ub ligase activity would seem contradictory if
the enhanced BRCA1 autoubiquitylation led to its degradation. It is now
clear that poly-Ub chains can be assembled through lysines other than
Lys48, and some of the resulting chains function in
distinct biological processes (47). In the most striking examples,
poly-Ub chains linked through Lys63 have been reported to
be involved in DNA repair (55-58), the stress response (59),
mitochondrial DNA inheritance (60), endocytosis (61), and ribosomal
function (62), suggesting that Lys63-linked Ub chains have
a signaling function that does not involve proteolysis. By carrying out
in vitro ubiquitylation assays using K48A and K63A Ub
mutants, we found that BARD1 stimulated the formation of both
Lys48- and Lys63-linked poly-Ub chains.
However, the enhancement of BRCA1 autoubiquitylation by BARD1 mostly
resulted in poly-Ub chains linked through Lys63 (Fig. 6).
The function of these Lys63-linked poly-Ub chains remains
to be determined but would be consistent with the proposed roles for
BRCA1 in DNA damage response and repair. While this paper was in
preparation, Chen et al. (33) also reported that BRCA1/BARD1
heterodimer could assemble non-Lys48-linked poly-Ub chains.
Now that we have observed that BARD1 enhances BRCA1 E3 Ub ligase
activity through a direct interaction in vitro, the key
questions are as follows. Does BRCA1/BARD1 function as an E3 ligase
in vivo, and what are the biological substrate(s) for this
activity? Some potential substrates have been suggested. For example,
activated Fanconi anemia protein FANCD2 colocalizes with BRCA1, and the absence of BRCA1 results in loss of IR-inducible FANCD2
monoubiquitylation (63). Since RNA polymerase II has been shown to be
ubiquitylated and degraded after DNA damage (64, 65), it is also
possible that polymerase II could be a substrate for BRCA1-BARD1 (66). BRCA1 has been reported to interact directly with the BRG1 subunit of
the SWI/SNF complex and to alter chromatin structure, suggesting that
BRCA1 may regulate various biological processes through a common
underlying mechanism, such as chromatin remodeling, which could involve
ubiquitylation of histones (11-14). Identification of the biological
substrate(s) of BRCA1 will be essential for an understanding of its
multiple functions in different biological processes.
The finding that the BRCA1/BARD1 RING finger complex promotes the
formation of Lys63-linked poly-Ub chains attached to BRCA1
raises another interesting question regarding how the specificity for
Lys63 linkage is achieved. Ubc4, Ubc5, Mms2/Ubc13, and
UEV1a/Ubc13 are involved in catalyzing the assembly of
Lys63 chains (58, 59, 67-69). Both the yeast Mms2/Ubc13
and human UEV1a/Ubc13 interact with their RING partner proteins Rad5
(56) and Traf6 (70), which may be either a cognate E3 or a substrate of
the respective E2/UEV heterodimers. It remains to be determined what
the Ub-conjugating enzyme(s) for BRCA1 E3 ligase activity are in
vivo (Ubc4 has been reported to catalyze formation of
Lys63 chains in yeast), how BRCA1 functions in the
recognition and assembly of Lys63 chains together with E2,
and what the possible function(s) of these BRCA1 attached
Lys63 chains are. Our future work will aim to elucidate the
functional mechanism of BRCA1 as an E3 Ub ligase and its role in tumor
suppression, which might provide new approaches for the treatment and
prevention of breast and ovarian cancers.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helices flanking the central RING motif of BRCA1
and BARD1 form a stable four-helix bundle that acts as the major
heterodimerization interface between the two proteins (15). Several
lines of evidence suggest that BARD1 is involved in BRCA1-mediated
tumor suppression. BARD1 mutations have been detected in breast,
ovarian, and uterine tumors (25), and inhibition of BARD1 expression in
cultured cells results in a premalignant phenotype (26). The
BRCA1-BARD1 complex has been shown to interact with the polyadenylation
factor CstF-50 (27), presumably to inhibit mRNA processing at sites
of DNA damage (28). BRCA1-BARD1 co-localize with DNA replication and
repair factors in response to DNA damage (29-32). Importantly, it has
been reported that BRCA1-BARD1 heterodimers exhibit significant E3 Ub
ligase activity and that the BARD1 RING finger domain greatly
potentiates the ligase activity of the BRCA1 RING finger (22, 33).
However, BARD1 may also have BRCA1-independent functions, since it can act as an apoptosis inducer in a BRCA1-independent manner (34).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-galactopyranoside for about 3 h
at 30 °C. The cell pellets were then lysed in lysis buffer (50 mM Tris-Cl, pH 8.0, 120 mM NaCl, 1 mM dithiothreitol, plus protease inhibitors). Proteins
bound to glutathione-agarose (Sigma) were eluted with
phosphate-buffered saline buffer containing 20 mM glutathione (pH 7.1-7.5) and dialyzed against 20 mM
Tris-Cl, pH 8.0, 50 mM NaCl, 10% glycerol, and 1 mM dithiothreitol. GST pull-down assays were performed as
described previously (38). Equal amounts of WT or mutant
GST-BARD1-(8-142) were mixed together with His-BRCA1-(1-110) on ice
for 30 min, in buffer containing 20 mM HEPES, pH 7.0, 1 mM EDTA, 10% glycerol, and protease inhibitors. The
mixtures were then incubated with glutathione-agarose for another 30 min with rolling at 4 °C, and the beads were washed extensively with
the same buffer supplemented with 150 mM NaCl and 0.1%
Nonidet P-40. Bound proteins were eluted with phosphate-buffered saline
buffer containing 20 mM glutathione (pH 7.1-7.5).
polyclonal antiserum from rabbit 5547.
-galactosidase (Novus Biologicals, Inc.) antibodies.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Enhancement of BRCA1 E3 ubiquitin ligase
activity by the BARD1 protein. A, a schematic
illustration of the BRCA1 (1863 amino acids) and BARD1 (777 amino
acids). Both maps show the N-terminal RING domain (BRCA1-(23-76),
BARD1-(49-100)) and C-terminal BRCT domains. B, in an
in vitro ubiquitylation assay, increasing amounts (0.25, 0.5, 0.75, and 1 µg) of purified GST-BRCA1-(1-110) were incubated
with purified His-E1, His-E2 (Ubc4), and GST-Ub in the absence of
GST-BARD1-(8-142) (lanes 3-6). The same amount
of BRCA1 (0.75 µg) as used in the lane marked with a
star was then incubated with increasing amounts (0.5, 1, and
2 µg) of purified GST-BARD1-(8-142) (lanes
7-9). Lane 1, a reaction containing
only E1 and E2; lane 2, a reaction containing
only BRCA1 besides GST-Ub. The membrane was probed with anti-Ub
antibody. Polyubiquitylated conjugates and GST-Ub are indicated on the
right. Molecular mass markers, in kDa, are shown on
the left. C, BRCA1 and BARD1 cooperate in the
monoubiquitylation of histone H2A. 0.25 µg of purified full-length
FLAG-tagged recombinant BRCA1 was incubated with 3 µg of
Drosophila core histones, His-E1, His-E2 (Ubc4), and GST-Ub
in the absence or presence of 0.5 µg of purified GST-BARD1-(8-142).
Lanes 1-6, negative controls in which the
reactions only contain the proteins indicated. Lanes
7 and 9, reactions containing BRCA1, E1, E2, Ub,
and histones in the absence or presence of BARD1, respectively.
Lane 8 shows that BARD1 cannot monoubiquitylate
H2A. The monoubiquitylated H2A is indicated on the right.
Molecular mass markers, in kDa, are shown on the left. The
membrane was probed with anti-Ub antibody (top) and anti-H2A
antibodies (bottom) separately. D, BARD1 RING
domain does not exhibit E3 ubiquitin ligase activity in
vitro. In an in vitro ubiquitylation assay, increasing
amounts (0.5, 1, 2, and 3 µg) of purified GST-BRCA1-(1-110)
(lanes 2-5) or increasing amounts (2, 4, 6, 8, and 12 µg) of purified GST-BARD1-(8-142) (lanes
6-10) were incubated with purified His-E1, His-E2 (Ubc4),
and GST-Ub. Lane 1 shows a reaction containing
only E1 and E2. The membrane was probed with anti-Ub antibody.
Polyubiquitylated conjugates and GST-Ub are indicated on the
right. Molecular mass markers, in kDa, are shown on the
left.
-helix, L44R (N-terminal
-helix) and I105D (C-terminal
-helix), which contain an intact RING domain but fail to bind BRCA1
in the yeast two-hybrid assay (15, 44). Consistently, the L44R mutant
protein showed no detectable binding, and the I105D mutant protein
showed only extremely weak binding to BRCA1 in the GST pull-down assay
(Fig. 2C).
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Fig. 2.
In vitro protein-protein
interaction between the RING finger domains of BRCA1 and wild type or
mutant BARD1. A, alignment of the RING domains of BRCA1
and BARD1. The conserved RING finger Cys and His residues are in
boldface type. The amino acid residues mutated in
this study are underlined, with glycine (G)
and/or alanine (A) marked above. B, 2 µg of purified His-BRCA1-(1-110) was incubated with 2 µg of
purified WT or mutant GST-BARD1-(8-142) and glutathione beads. After
extensive washing, the bound proteins were eluted with
phosphate-buffered saline buffer containing 20 mM
glutathione and then boiled in SDS sample buffer for analysis. The
membrane was immunoblotted with anti-(His)5 monoclonal
antibody. In lane 1, the same amount of purified
GST was used instead of BARD1 as a negative control. In lane
11, one-tenth the amount of His-BRCA1 (1-110) was loaded as
a positive control. His-BRCA1-(1-110) is indicated on the
right, and the molecular mass markers are shown on the
left. C, 2 µg of purified His-BRCA1-(1-110)
was incubated with 2 µg of purified WT or mutant GST-BARD1-(8-142)
(L44R or I105D) and glutathione beads. The membrane was immunoblotted
with anti-BRCA1 (A) antibodies. In lane 4,
one-tenth amount of His-BRCA1-(1-110) was loaded as a positive
control. His-BRCA1-(1-110) is indicated on the right, and
the molecular weight markers are shown on the left.
-helix (residues 81-96) flanking the central RING motif
(15), consistent with the previous report that BARD1 does not interact
stably with the N-terminal 71 residues of BRCA1 (23) and the fact that
this helix is critical for the four-helix bundle (15). These results indicate that a direct stable interaction between BARD1 and BRCA1 is
required for BARD1 to enhance BRCA1 E3 Ub ligase activity and that the
integrity of the BARD1 RING domain is not so important.
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Fig. 3.
The effects of wild type or mutant BARD1 RING
domain on BRCA1 E3 ubiquitin ligase activity. 0.5 µg of purified
GST-BRCA1-(1-110) or GST-BRCA1-(1-78) were incubated with purified
His-E1, His-E2 (Ubc4), and GST-Ub, in the absence or presence of 1 µg
of purified WT or mutant GST-BARD1-(8-142) (lanes
3-16). Lane 1 shows a reaction
containing only E1 and E2, whereas lane 2 shows a
reaction containing only BRCA1-(1-110) and BARD1, in addition to
GST-Ub. The reactions were separated by SDS-PAGE and analyzed by
immunoblotting with anti-GST antibody. GST-Ub and polyubiquitylated
conjugates are indicated on the right, and the molecular
mass markers, in kDa, are shown on the left. The reactions
were done in parallel (lanes 1-9, 15,
and 16) or separately with controls (lanes
10-14).
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Fig. 4.
BRCA1 and BARD1 control each other's
abundance in living cells. A, elevation of
BRCA1-(1-308) levels by BARD1. 293T cells were transfected with 10 µg of FLAG-BRCA1-(1-308) plasmid and 10 µg of FLAG plasmid
(lane 1) or with 2, 6, or 10 µg of
FLAG-BARD1-(8-142) plasmid (lanes 2-4) or with
2, 6, or 10 µg of FLAG-BARD1 (full-length) plasmid (lanes
5-7). Vector plasmid pFLAG was added to maintain equivalent
plasmid DNA concentrations in each transfection. The membrane was
analyzed by immunoblotting with anti-FLAG antibody. Protein bands for
FLAG-BRCA1-(1-308), FLAG-BARD1-(8-142) and FLAG-BARD1 (full-length)
are indicated. Equal loading of cell lysates was confirmed by probing
the same membrane with anti-GST-Nck- antiserium. B,
elevation of full-length BRCA1 level by BARD1. 293T cells were
co-transfected with 10 µg of pHIV CSC BRCA1 plasmid and 10 µg of
pBluescript II (KS+) control plasmid or with 10 µg of pHIV CSC BRCA1
plasmid and 10 µg of FLAG-BARD1 (full-length) plasmid. 4 µg of pCMX
LacZ and 1 µg of pEGFP C-2 were transfected as controls for
transfection efficiency (data not shown). The membrane was probed with
the anti-BRCA1 MS110 monoclonal antibody. The values below
each lane are the relative amounts of BRCA1 after
normalization to the transfection efficiency (BRCA1
level/
-galactosidase level). BRCA1 and
-galactosidase signals
were quantitated with the NIH Image software package, and the relative
BRCA1 level from 293T cells transfected with BRCA1 and vector control
was set as 1. C, the elevation of BRCA1 levels by BARD1 is
dependent on the direct interaction between the two proteins. 293T
cells were co-transfected with 10 µg of FLAG-BRCA1-(1-308) plasmid
and 10 µg of pFLAG or with 10 µg of WT or mutant FLAG-BARD1
(full-length) plasmid. The FLAG-BRCA1-(1-308) band is indicated. Equal
loading of cell lysates was confirmed by probing the same membrane with
anti GST-Nck-
antiserum. D, elevation of BARD1 levels by
BRCA1. 293T cells were transfected with 10 µg FLAG-BARD1-(8-142)
plasmid and 10 µg of pFLAG plasmid (lane 1) or
with 2, 6, or 10 µg of FLAG-BRCA1-(1-308) plasmid (lanes
2-4). 10 µg of FLAG-BARD1 (full-length) plasmid was
co-transfected with 10 µg of pFLAG (lane 5) or
with 2, 6, or 10 µg of FLAG-BRCA1-(1-308) plasmid (lanes
6-8). Vector plasmid pFLAG was added to maintain equivalent
plasmid DNA concentrations in each transfection. FLAG-BRCA1-(1-308),
FLAG-BARD1-(8-142), and FLAG-BARD1 (full-length) are indicated. Equal
loading of cell lysates was confirmed by probing the same membrane with
anti-GST-Nck-
antiserum.
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Fig. 5.
BRCA1 and BARD1 stabilize each other in
living cells through direct interaction. 293T cells were
co-transfected with 10 µg of full-length FLAG-BARD1 (WT) plasmid and
10 µg of pFLAG plasmid (lanes 1-5); with 10 µg of full-length FLAG-BARD1 (C50G/H68A) plasmid and 10 µg of WT
FLAG-BRCA1-(1-308) plasmid (lanes 6-10); with
10 µg of full-length FLAG-BARD1 (WT) plasmid and 10 µg of WT
FLAG-BRCA1-(1-308) plasmid (lanes 11-15); or
with 10 µg of WT FLAG-BRCA1-(1-308) plasmid and 10 µg of pFLAG
plasmid (lanes 16-20). 48 h later, the
cells were treated with the protein synthesis inhibitor cycloheximide
(150 µg/ml final concentration) for the times indicated. Cell lysates
were subjected to immunoblot analysis with anti-FLAG antibody.
FLAG-BRCA1-(1-308) and FLAG-BARD1 (full-length) are indicated. Equal
loading of cell lysates was confirmed by probing the same membranes
with an anti-GST-Nck- antiserum.
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Fig. 6.
In vitro ubiquitylation of BRCA1
with WT or mutant GST-Ub in the absence or presence of BARD1. The
same amount (1 µg) of purified GST-BRCA1-(1-110) was incubated with
purified His-E1, His-E2 (Ubc4), and equal amounts (10 µM)
of purified WT or the indicated mutant GST-Ub in the absence or
presence of 2 µg of purified GST-BARD1-(8-142). The first
lane, in which no BRCA1 or BARD1 was added, and the
second lane, in which only BRCA1 and BARD1 were
added, represent negative controls. The membrane was probed with
anti-BRCA1 (A) antibodies and then reprobed with anti-Ub antibody after
stripping. GST-Ub and GST-BRCA1-(1-110) are indicated.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helix in its RING domain that is important for the c-Cbl RING (18) and Rbx1 RING
(51) to interact with E2, which may explain its lack of E3 ligase
activity. Our in vitro studies show that the BARD1 RING
domain and surrounding helices have a different function, namely to
enhance the BRCA1 E3 activity. In support of this, we found that both
autoubiquitylation of BRCA1-(1-110) and monoubiquitylation of histone
H2A by full-length BRCA1 were significantly enhanced by BARD1 (Fig. 1,
B and C), consistent with the recent report from
Pan's group (33). Our results could explain the Ohta group's finding
that BARD1 immunocomplexes have E3 ligase activity, since the
transfected Myc-BARD1 may have formed a complex with endogenous BRCA1
and stimulated its activity (22).
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ACKNOWLEDGEMENTS |
---|
We thank Jill Meisenhelder for the
anti-GST-Nck- antiserum, Dr. Richard Baer for the GST-BARD1
construct, and Joaquin Espinosa and Beverly Emerson for
Drosophila core histones.
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FOOTNOTES |
---|
* This work was supported by NCI, National Institutes of Health, United States Public Health Service Grants CA14195, CA80100, and CA82683 (to T. H.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Supported by funds provided by the Breast Cancer Research Program, Grant Number 8FB-0124.
** An American Cancer Society Professor of Molecular Biology.
A Frank and Else Schilling American Cancer Society Research
Professor. To whom correspondence should be addressed: 10010 N. Torrey
Pines Rd., La Jolla, CA 92037. Tel.: 858-453-4100 (ext. 1385); Fax:
858-457-4765; E-mail: hunter@salk.edu.
Published, JBC Papers in Press, November 12, 2002, DOI 10.1074/jbc.M204591200
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ABBREVIATIONS |
---|
The abbreviations used are: E2, ubiquitin conjugating enzyme; E1, ubiquitin activating enzyme; E3, ubiquitin-protein isopeptide ligase; Ub, ubiquitin; WT, wild type; aa, amino acid(s); GST, glutathione S-transferase.
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