From the Division of Breast and Endocrine Surgery, St. Marianna University School of Medicine, Kawasaki, 216-8511 Japan
Received for publication, December 14, 2000, and in revised form, February 20, 2001
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ABSTRACT |
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BRCA1-BARD1 constitutes a
heterodimeric RING finger complex associated through its N-terminal
regions. Here we demonstrate that the BRCA1-BARD1 heterodimeric RING
finger complex contains significant ubiquitin ligase activity that can
be disrupted by a breast cancer-derived RING finger mutation in
BRCA1. Whereas individually BRCA1 and BARD1 have very low ubiquitin
ligase activities in vitro, BRCA1 combined with BARD1
exhibits dramatically higher activity. Bacterially purified RING finger
domains comprising residues 1-304 of BRCA1 and residues 25-189 of
BARD1 are capable of polymerizing ubiquitin. The steady-state level of
transfected BRCA1 in vivo was increased by co-transfection
of BARD1, and reciprocally that of transfected BARD1 was increased by
BRCA1 in a dose-dependent manner. The breast cancer-derived
BARD1-interaction-deficient mutant, BRCA1C61G, does not
exhibit ubiquitin ligase activity in vitro. These
results suggest that the BRCA1-BARD1 complex contains a ubiquitin
ligase activity that is important in prevention of breast and ovarian cancer development.
Germline mutations of BRCA1 predispose women to breast and ovarian
cancers (1). BRCA1 contains several domains that interact with a
variety of molecules and is potentially responsible for multiple
functions in DNA damage repair, transcription, and cell-cycle regulation (2-4). BARD1 was identified in a yeast two-hybrid screen as
a protein that interacts with BRCA1 (5). Both BRCA1 and BARD1 proteins
contain a RING finger (5) and exist as homodimers or preferentially
form stable heterodimers (6). The heterodimeric interaction is mediated
by the flanking regions of the RING finger motif of the two molecules
(6). Although a transcriptional function in the C terminus of BRCA1 has
been recently reported (3), the biochemical function of the
heterodimeric RING finger constituted from the N termini of BRCA1 and
BARD1 is not known.
Previously, we and others identified a highly conserved small RING
finger protein, ROC1 (also called Rbx1 and Hrt1), as an essential
subunit of the SCF Ub1 ligase
(7-10). The Ub ligase (E3) catalyzes the formation of polyubiquitin
chains onto substrate proteins via isopeptide bonds utilizing the Ubs
that have been sequentially activated by enzymes E1 and E2.
Polyubiquitinated substrates are then rapidly degraded by the 26 S
proteasome (11). The SCF and the APC are the two major Ub ligase
complexes that regulate ubiquitin-mediated proteolysis during
G1/S and anaphase (12), and contain the small RING finger proteins ROC1 and APC11, respectively (7-10). Point mutations in the
RING finger domain of ROC1 completely disrupted the Ub ligase activity,
suggesting an essential role of the domain for its activity (7). APC11
also contains Ub ligase activity in vitro (7). More
recently, several large RING finger proteins, such as MDM2, c-Cbl, IAP,
and AO7, with otherwise diverse structures and functions were linked to
ubiquitination (13-16), suggesting a potentially broad and general
function for RING fingers in activating Ub ligase activity. One of
these RING proteins, BRCA1, has been closely scrutinized for Ub ligase
activity. However, the ability of BRCA1 by itself to promote ubiquitin
polymerization was found to be limited (16).
In this report, we have provided evidence demonstrating that the RING
finger of BRCA1, in concert with BARD1, exhibits significant ubiquitin
ligase activity. This activity can be disrupted by a breast
cancer-derived RING finger mutation of BRCA1, suggesting a direct
relationship between the ubiquitin ligase function of BRCA1 and breast
cancer development.
Plasmids--
cDNAs for full-length human BARD1 and CstF-50
were amplified by polymerase chain reaction from a Hela cell cDNA
library with pfu polymerase (Stratagene) and subcloned into the
pcDNA3 vector inframe with appropriate tags.
pGEX-BARD1-(1-111) was created by digesting the full-length
BARD1 with HindIII, and subcloning into the pGEX-KG vector.
pET-His6-BARD1-(14-189) and
pET-His6-BARD1-(25-189) were created by digesting the
full-length BARD1 with BamHI and PstI, and
NcoI and PstI, respectively and subcloned into
the pET-3E-His6 vector. Full-length BRCA1 cDNA was a
generous gift from Dr. Wen-Hwa Lee. The N-terminal fragment (1) of
BRCA1 was generated by digesting the full-length BRCA1 at its
KpnI restriction site and subcloning into either the pGEX
vector or the pcDNA3 vector with the appropriate tag.
pGEX-BRCA1-(1-342) was created by self-ligation of
BglII-digested pGEX-BRCA1-(1-772).
pET-His6-BRCA1-(1-304) was created by digesting BRCA1-(1-772) with EcoRI and subcloning into the
pET-3E-His6 vector. BRCA1 or BARD1 point mutations were
introduced by site-directed mutagenesis (Stratagene). cDNA for each
human E2/Ubc was amplified by polymerase chain reaction from a HeLa
cell cDNA library and subcloned into the pET-3E-His6
vector. cDNA for human E2F1 and cyclin B1 in the pcDNA3
expression vector were gifts from Dr. Yue Xiong. All the constructs
used were verified by DNA sequencing.
Cell Culture, Transfection, and Immunoprecipitation--
Cells
(293T) were cultured in Dulbecco's modified Eagle's medium (Sigma)
supplemented with 10% fetal bovine serum (Life Technologies, Inc.) and
1% antibiotic-antimycotic agent (Life Technologies, Inc.) in a
37 °C incubator with 5% CO2. DNA was transfected using the standard calcium phosphate precipitation method. For each transfection, the total plasmid DNA was adjusted to 15 µg per 100-mm
dish by adding the parental pcDNA3 vector. For immunoprecipitation, cells were harvested 36 h after transfection and lysed by
incubating at 4 °C for 1 h with 0.6 ml per 100 mm dish of
buffer A containing 15 mM Tris-HCl pH 7.5, 0.5 M NaCl, 0.35% Nonidet P-40, 1 mM
phenylmethylsulfonyl fluoride, 2 µg/ml aprotinin, 2 µg/ml
leupeptin, 10 µg/ml trypsin inhibitor, and 150 µg/ml benzamidine.
Lysed cells were then clarified by centrifugation at 100,000 × g at 4 °C for 1 h. The supernatants (0.3 ml) were
mixed with 3 µg of anti-Myc (9E10) or anti-HA (12CA5) antibody, and then the antibody-bound proteins were precipitated with
protein A-agarose beads (7.5 µl). The proteins bound to the beads
were used either for the Ub ligation assay or immunoblotting. For
straight immunoblotting to analyze the steady-state levels of Myc-BRCA1
or HA-BARD1, the transfected cells were lysed and clarified as
described above, and 50 µg of each sample was resolved by SDS-PAGE on
a 7.5% gel, followed by immunoblotting.
Recombinant Proteins--
Rabbit E1 was purchased from Affiniti
Research Products. His6-tagged Ub with a protein kinase C
recognition site, His6-tagged E2 proteins, and
His6-E2F1 were purified as previously described (7, 17).
His6-BARD1-(14-189), His6-BARD1-(25-189),
His6-BRCA1-(1-304), GST-BRCA1-(1-342) and GST-BARD1-(1-111)
were produced in BL21/DE3 bacteria by induction with 0.4 mM
IPTG for 12 h at 25 °C. Cells were lysed in buffer containing 50 mM Tris-HCl, pH 8.0, 0.5% NP-40, 1% Triton X-100, 50 mM NaCl, 1 mM DTT, 1 mM EDTA, 10%
glycerol, and protease inhibitors, and the proteins were purified
either with nickel beads (Qiagen) or glutathione-agarose beads (Sigma) according to the manufacturer's instructions.
Ub Ligation Assay--
The procedure for the in vitro
Ub ligation assay was essentially the same as previously described (7,
18). The BRCA1-BARD1 immunocomplexes immobilized on protein A-agarose
beads were washed three times with buffer A and two times with buffer B
containing 25 mM Tris-HCl, pH 7.5, 50 mM NaCl,
0.01% Nonidet P-40, 10% glycerol, and 1 mM EDTA, and
added to a ubiquitin ligation reaction mixture (30 µl) that contained
50 mM Tris-HCl, pH 7.4, 5 mM MgCl2,
2 mM NaF, 10 nM okadaic acid, 2 mM
ATP, 0.6 mM DTT, 0.75 µg of 32P-Ub, 40 ng of
E1, and 0.3 µg of E2 protein. After incubation for 30 min at 37 °C
with shaking, the reactions were terminated by boiling in Laemmli
SDS-loading buffer with 0.1 M DTT, and half of the sample
was resolved by SDS-PAGE followed by autoradiography. For the in
vitro Ub ligation assay using the purified RING finger domain of
BRCA1 or BARD1, GST-fused proteins bound to glutathione-agarose beads,
and eluted His6-tagged proteins were used instead of
immunocomplexes. The activity of E2 was analyzed by the same procedure
without E3.
Ub Ligase Activities of the BRCA1 or BARD1 Immunocomplexes in
Collaboration with E2/UbcH5c--
The discovery that the small RING
finger proteins, ROC1 and ROC2, are ubiquitin ligases prompted us to
determine whether BRCA1 and BARD1, two of the most important RING
finger proteins implicated in breast cancer, also function as ubiquitin
ligases. We first determined which E2/Ubc could be activated by BRCA1
or BARD1. Five representative mammalian E2s (UbcH1, UbcH2, Cdc34,
UbcH5c and UbcH7) were purified from bacteria (Fig.
1A). Individual E2s were
incubated with E1 and 32P-Ub in the presence of ATP to
verify their Ub binding capacity. Four of the five E2s (UbcH2, Cdc34,
UbcH5c, and UbcH7) were determined to be active enzymes by judging the
ability to bind to 32P-Ub (Fig. 1B, lanes
3-6, lower panel), and was dissociated by addition of
DTT (lanes 3-6, upper panel). When the
anti-Myc immunocomplex derived from Myc-BRCA1-(1-772) or
Myc-BARD1-transfected 293T cells was added to the reaction, only UbcH5c
among the five E2s was capable of promoting ubiquitin polymerization
with the immunocomplex as determined by the appearance of a high
molecular weight 32P smear (Fig. 1C, lanes
4 and 9). Although a crystal structure study revealed
the interaction between the RING finger of c-Cbl and UbcH7 (19), UbcH7
was deficient in collaborating with the RING finger of BRCA1 or BARD1
to promote Ub polymerization (lanes 5 and 10).
These results suggest that the BRCA1- and BARD1-associated protein
complexes contain Ub ligase activities.
Ub Ligase Activity of the BRCA1-BARD1 Immunocomplex and Its
Inactivation by a RING Finger Mutation of BRCA1--
We noted that the
Ub ligase activities of the BRCA1 and BARD1 immunocomplexes, which were
only detectable after long exposure of the film (Fig. 1C),
were very low and sought conditions for higher activity. Because it was
reported that BRCA1 and BARD1 proteins preferentially formed stable
heterodimers (6), we next tested whether the BRCA1-BARD1 heterodimer
complex formation affects Ub ligase activity. Surprisingly, when
Myc-BRCA1-(1-772) was co-transfected with HA-BARD1, the anti-Myc
immunocomplex exhibited significantly higher Ub ligase activity (Fig.
2A, lane 5) than that of either the Myc-BARD1 (lane 3) or Myc-BRCA1-(1-772)
(lane 4) single transfection. It is possible that the low Ub
ligase activity detected with the immunocomplexes from single
transfections of BRCA1 or BARD1 may be caused by contamination with the
endogenous partner RING finger protein. Similarly, addition of
Myc-BRCA1-(1-772) dramatically enhanced the Ub ligase activity of the
HA-BARD1 immunocomplex (lane 8). E2F1 and cyclin B1, the
proteins known to interact with the N terminus of BRCA1 (20), as well
as CstF50, a protein that binds to BARD1 (21) do not have a stimulatory
effect on Ub ligase activity of BRCA1 or BARD1 (Fig. 2B,
lanes 1-6), arguing against the possibility that the
stimulatory effect observed with BRCA1-BARD1 is mediated by stabilizing
the structure of each protein by its partners nonspecifically. Coupled
immunoprecipitation and Western blotting verified the interactions
between these proteins (lane 7). To verify the importance of
the RING finger in activity and to explore the relationship between the
BRCA1 tumor suppressor function and the Ub ligase activity, we made
constructs containing BRCA1 with a tumor-derived mutation at Cys-61
(BRCA1C61G) (22). The mutation completely abolished Ub
ligase activity (Fig. 2C, lane 3) when compared
with the anti-Myc immunocomplex precipitated from cells cotransfected
with wild-type Myc-BRCA1-(1-772) and HA-BARD1 (lane 2) and
reduced the activity of the HA-BARD1 immunocomplex to the level of the
corresponding HA-BARD1 single transfection (lanes 7 versus 5). An artificial mutation in the RING
finger domain, BRCA1C39A/H41A, also disrupted the activity
to the same degree as BRCA1C61G (lanes 4 versus 3 and 8 versus 7). On the
other hand, BARD1C83G and BARD1C66A/H68A, the
RING mutations corresponding to those of BRCA1C61G and
BRCA1C39A/H41A, respectively, did not abolish the Ub ligase
activity, although a detectable reduction of the activity was observed
(Fig. 2D, lanes 3 and 4).
Ub Ligase Activity of the Purified RING Finger Domains of BRCA1 and
BARD1--
To exclude the possibility that the ligase activity in the
immunocomplex is caused by contaminating proteins and also to determine the core of the Ub ligase activity, both the RING finger domain of
BRCA1 and of BARD1 were purified from bacteria via GST or
His6 tags (Fig.
3A). Consistent with the
immunocomplex-based assay, a mixture of the purified RING finger
domains of BRCA1 and BARD1 exhibited significant Ub ligase activity
(Fig. 3B, lanes 3, 6, 8, and 10)
whereas either the BRCA1 (lanes 1 and 5) or BARD1
(lanes 2 and 7) RING alone displayed barely
detectable Ub ligase activity. Purified His6-E2F1 did not
enhance the Ub ligase activity of BRCA1 (lane 9), although
it did interact with GST-BRCA1-(1-342) (data not shown). RING finger
domains comprising residues 1-304 of BRCA1 and residues 25-189 of
BARD1 were capable of polymerizing ubiquitin (lane 8).
Importantly BRCA1C61G, the breast cancer-derived RING
finger mutant, did not collaborate with BARD1 to activate UbcH5c
(lane 4). Taken together, these results indicate that BARD1
and BRCA1 collaboratively activate Ub ligase activity with the E2
UbcH5c. The reactants contained ubiquitinated products migrating at
~35 and 43 kDa (Fig. 3, BRCA1 and BARD1 Stabilize Each Other in Vivo--
There is a
possibility that these two molecules are ubiquitination substrates of
each other for targeted degradation. We therefore analyzed the
steady-state levels of these proteins by Western blotting.
Polyubiquitinated substrates are rapidly degraded by the 26 S
proteasome and, correspondingly, the steady-state level of such
substrates tends to drop detectably. However, the steady-state level of
Myc-BRCA1-(1-772) was increased after co-transfection of HA-BARD1 in a
dosage-dependent manner (Fig.
4A). Reciprocally, the
steady-state level of HA-BARD1 was increased when it was cotransfected with Myc-BRCA1-(1-772) (Fig. 4B). These results indicate that BRCA1
and BARD1 stabilized each other while gaining the ability to ligate the
polyubiquitin chain, suggesting that they are not substrates signaled
by each other for degradation.
The RING finger motif was thought to be a DNA binding site when
BRCA1 was first identified as a tumor suppressor gene for familial
breast and ovarian cancer, because it was related to the zinc finger, a
known DNA binding motif (1). Moreover, the involvement of the BRCT
domain in transcription suggests that BRCA1 is a transcription factor,
and the RING finger takes part in DNA binding. However, despite
tremendous effort, binding between the RING finger of BRCA1 and DNA has
not been shown. Instead, the RING finger of BRCA1 has been shown to be
important for a protein-protein interaction with another RING finger
domain, which was identified and given the name BARD1 (5). A detailed
biochemical analysis revealed, however, that the interaction between
BRCA1 and BARD1 was mediated by regions outside the canonical RING
finger motif (6), suggesting that the two RING fingers contained some additional function where the proximity of the two RINGs was important. In the present report, we provide evidence demonstrating that the RING
heterodimer BRCA1-BARD1 functions as a Ub ligase. First, the
BRCA1-BARD1 immunocomplex obtained from BRCA1- and BARD1-transfected cells contains a significant ability to promote polyubiquitination in vitro, and omission of either one of the two from the
transfection eliminates this activity. Next, the bacterially purified
RING finger domains of BRCA1 and BARD1 exhibit high Ub ligase activity. Again omission of either one of the two RING fingers eliminates this
activity. Most importantly, in both experiments, a breast cancer-derived RING finger mutation of BRCA1, C61G, abolishes the
ability to promote polyubiquitination. Although it is possible that the
major reaction being monitored in the present study is autoubiquitination, BRCA1 and BARD1 are not substrates signaled by each
other for degradation, because they stabilize each other in
vivo. A common feature of RING finger ubiquitin ligases is autoubiquitination (Refs. 13-16 and 18). The importance of those autoubiquitinations in the function of RING type ubiquitin ligases remains to be determined.
BRCA1-BARD1 may constitute a novel type of RING containing Ub ligase
that forms a heterodimeric RING finger complex. However, the
stoichiometry of RING finger molecules in complexes of known RING Ub
ligases has not yet been elucidated. It remains to be determined
whether dimerization, either as homo- or heterodimers, between two
RING fingers represents a general mechanism for activating Ub ligase
activity. Supporting this possibility, Myc-ROC1 can be detected in
HA-ROC1 immunocomplexes precipitated from cells transfected with
Myc-ROC1 and HA-ROC1.2
During the S phase of the cell cycle, the steady-state levels of BRCA1
reach a maximum, and BARD1 colocalizes with BRCA1 into nuclear dots
(23). Consequently it is likely that an in vivo Ub ligase
activity of BRCA1-BARD1 would reach a maximum in S phase. Whether the
formation of the heterodimer is a regulated event possibly resulting in
the stabilization of BRCA1 and in vivo Ub ligase activity
remains to be determined. To further understand the biological function
of the BRCA1-BARD1 Ub ligase, determining the substrates presumably
targeted for degradation is critical. Nuclear proteins that are
degraded or down-regulated in S phase may be candidate substrates for
the BRCA1-BARD1 Ub ligase. The high Ub ligase activity of BRCA1-BARD1
presented here should make identifying those candidates easier.
The fact that the two RING finger domains of BRCA1 and BARD1 are
necessary for one biochemical function indicates that the oncogenic
potential or the phenotypes caused by mutations in the domains of those
two molecules should be the same. Indeed, germline mutations in the
BARD1 gene in primary breast and ovarian cancers have
been reported (24). BARD1 is potentially as important a gene
as BRCA1 in tumor suppression of breast and ovarian cancers. By correlating each mutation of the two molecules and BRCA1-BARD1 in vitro Ub ligase activity, it may now be possible to
predict the oncogenic potential for breast and ovarian cancer in
families with germline missense point mutations in the RING finger
domains of BRCA1 or BARD1. As such, the finding that the BRCA1-BARD1
heterodimer complex acts as a Ub ligase should provide a new insight to
further clinical research into the roles of BRCA1 and BARD1 in tumor suppression.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
E2/UbcH5c collaborates with the Myc-BRCA1 or
Myc-BARD1 immunocomplex to promote Ub polymerization.
A, individual purified His6-tagged E2 proteins
(2 µg) were resolved by SDS-PAGE followed by Coomassie Blue staining.
B, the purified E2s were mixed with E1,
32P-labeled Ub, and ATP. After incubation, the reactions
were terminated by boiling in Laemmli SDS loading buffer with
(upper panel) or without (lower panel) 0.1 M DTT and were resolved by SDS-PAGE followed by
autoradiography. C, cell lysates from 293T cells transfected
with Myc-BRCA1-(1-772) (left panel) or Myc-BARD1
(right panel) were immunoprecipitated with anti-Myc
antibody. Immunocomplexes immobilized on the beads were subjected to Ub
ligation assay with the indicated EZ (overnight exposure).
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Fig. 2.
In vitro Ub ligase activity of the
BRCA1-BARD1 immunocomplex. A-D, cell lysates were
obtained from 293T cells transfected with 7.5 µg of plasmid indicated
at the top of each panel, followed by
immunoprecipitation (IP). A substrate-nonspecific Ub
ligation assay with E2/UbcH5c was performed (30-min exposure). The
BRCA1 and BARD1 proteins in the immunocomplexes were verified by
immunoblotting either with anti-Myc or anti-HA antibody (A,
C, and D; bottom panel). The
interaction between Myc-BRCA1 and either E2F1 or cyclin B1, or between
Myc-BARD1 and HA-CstF50 was verified by coupled anti-Myc
immunoprecipitation and immunoblotting with anti-E2F1, anti-cyclin B1,
or anti-HA, respectively (B, right panel). Note
that the activity of the Myc-BARD1 immunocomplex in lane 3 in A corresponds to that in lane 9 in Fig.
1C. and
at the right of the
panel indicate the positions of Myc-BRCA1-(1-772) and
HA-BARD1 migration, respectively.
), exactly the same position as that seen
in the immunocomplex-based assay (Fig. 2). These products are likely to
be free ubiquitin polymers or ubiquitin polymers covalently bound to
some small molecule such as UbcH5c. The majority of the
polyubiquitinated products including these smaller products did not
dissociate from the BRCA1-BARD1 complex when washed with buffer B (data
not shown).
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Fig. 3.
In vitro Ub ligase activity of the
bacterially purified RING finger domains of BRCA1 and BARD1.
A, GST or His6-tagged RING finger domains of
BRCA1 and BARD1 were purified from bacteria, and each protein (5 µg)
was resolved by SDS-PAGE followed by Coomassie Blue staining.
B, the indicated amounts (µg) of the GST-fused RING finger
domains of BRCA1 or BARD1 bound to glutathione-agarose beads and the
His6-tagged RING finger domains of BRCA1 or BARD1 eluted in
phosphate-buffered saline were mixed. The mixtures were subjected to UB
ligation assay with Ez/UbcH5c.
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Fig. 4.
Stabilization of BRCA1 and BARD1 by their
partner RING finger in vivo. A, cell
lysates from 293T cells transfected with plasmids expressing
Myc-BRCA1-(1-772) (lanes 1-4, 7.5 µg), and HA-BARD1
(lane 2, 0.2 µg; lane 3, 1.0 µg; lane
4, 7.5 µg) were subjected to anti-Myc or anti-HA immunoblotting.
B, the steady-state level of Myc-BRCA1-(1-772) and HA-BARD1
was analyzed as described in A from the cells transfected
with plasmids expressing Myc-BRCA1-(1-772) (lane 2, 0.5 µg; lane 3, 3.0 µg; lane 4, 14 µg) and
HA-BARD1 (lanes 1-4, 1.0 µg) as indicated.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank Dr. Yue Xiong and Christopher W. Jenkins for helpful discussions and critical reading of the manuscripts, Dr. W-H. Lee for kindly providing BRCA1 cDNA. We also thank Takako Kuwahara for secretarial assistance.
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FOOTNOTES |
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* This study was supported by a grant-in-aid for general scientific research from the Ministry of Education, Science, Sports, and Culture of Japan, and by a grant-in-aid from the Tokyo Biochemical Research Foundation.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.
To whom correspondence should be addressed. Tel.: 81-44-977-8111;
Fax: 81-44-976-5964; E-mail: to@marianna-u.ac.jp.
Published, JBC Papers in Press, March 6, 2001, DOI 10.1074/jbc.C000881200
2 T. Ohta, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are:
Ub, ubiquitin;
E1, ubiquitin-activating enzyme;
E2, ubiquitin carrier protein;
SCF, SKP1-CUL1 (CDC53)- F-box protein complex;
APC, anaphase-promoting
complex;
PAGE, polyacrylamide gel electrophoresis, GST, glutathione
S-transferase;
HA, hemagglutinin;
DTT, dithiothreitol;
IPTG, isopropyl-1-thio--D-galactopyranoside.
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