[Cancer Research 65, 6-10, January 1, 2005]
© 2005 American Association for Cancer Research
Down-regulation of BRCA1-BARD1 Ubiquitin Ligase by CDK2
Ryosuke Hayami1,
Ko Sato1,
Wenwen Wu1,
Toru Nishikawa1,
Junya Hiroi2,
Ritsuko Ohtani-Kaneko2,
Mamoru Fukuda1 and
Tomohiko Ohta1
1 Division of Breast and Endocrine Surgery, and 2 Department of Anatomy, St. Marianna University School of Medicine, Kawasaki, Japan
Requests for reprints: Tomohiko Ohta, Division of Breast and Endocrine Surgery, Department of Surgery, St. Marianna University School of Medicine, Kawasaki, 216-8511 Japan. Phone: 81-44-977-8111; Fax: 81-44-976-5964. E-mail: to{at}marianna-u.ac.jp.
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Abstract
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BRCA1, a breast and ovarian tumor suppressor, is a phosphoprotein whose cellular expression level is regulated in a cell cycle–dependent manner. BRCA1 interacts with BARD1 to generate significant ubiquitin ligase activity which catalyzes nontraditional Lys-6-linked polyubiquitin chains. However, it is not clear how the activity is regulated and how this affects BRCA1's multiple cellular functions. Here we show that the ubiquitin ligase activity of BRCA1-BARD1 is down-regulated by CDK2. During the cell cycle, BARD1 expression can largely be categorized into three patterns: moderately expressed in a predominantly unphosphorylated form in early G1 phase, expressed at low levels in both phosphorylated and unphosphorylated forms during late G1 and S phases, and highly expressed in its phosphorylated form during mitosis coinciding with BRCA1 expression. CDK2-cyclin A1/E1 and CDK1-cyclin B1 phosphorylate BARD1 on its NH2 terminus in vivo and in vitro. Intriguingly, the BRCA1-BARD1–mediated in vivo ubiquitination of nucleophosmin/B23 (NPM) and autoubiquitination of BRCA1 are dramatically disrupted by coexpression of CDK2-cyclin A1/E1, but not by CDK1-cyclin B1. The inhibition of ubiquitin ligase activity is not due to the direct effect of the kinases on BARD1 because an unphosphorylatable mutant of BARD1, S148A/S251A/S288A/T299A, is still inhibited by CDK2-cyclin E1. Alternatively, BRCA1 and BARD1 are likely exported to the cytoplasm and their expressions are remarkably reduced by CDK2-cyclin E1 coexpression. Recognizing the importance of cyclin E1 overexpression in breast cancer development, these results suggest a CDK2-BRCA1-NPM pathway that coordinately functions in cell growth and tumor progression pathways.
Key Words: BARD1 • BRCA1 • CDK2 • Nucleophosmin • Ubiquitin
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Introduction
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BRCA1 has been implicated in an amazingly diverse range of biological processes, such as DNA repair, cell cycle control, transcriptional regulation, apoptosis, and centrosome duplication (1). Furthermore, BRCA1 acquires significant ubiquitin ligase activity when bound to BARD1 as a RING heterodimer (2). The most commonly catalyzed polyubiquitin chain is linked through Lys-48 of ubiquitin and serves as a signal for rapid degradation of substrates by the proteasome-dependent proteolysis pathway. In contrast, BRCA1-BARD1 catalyzes Lys-6-linked polyubiquitin chains (3–5), which are recognized in vitro by the 26S proteasome for deubiquitination as opposed to degradation (4). These observations suggest that ubiquitination mediated by BRCA1-BARD1 could signal a process other than degradation. However, events both upstream and downstream of the ligase activity remain to be determined. As one possible key downstream event, we recently showed that the BRCA1-BARD1 heterodimer catalyzes the polyubiquitination of nucleophosmin/B23 (NPM; ref.6). NPMisan acidic nucleolar phosphoprotein that is known tobe important in multiple cellular functions including ribosomal biogenesis, cell proliferation and centrosome duplication (7, 8). Here, we report a possible upstream event in which CDK2 down-regulates the ubiquitin ligase activity of BRCA1-BARD1. These results suggest cell cycle–dependent control of BRCA1-BARD1 ubiquitin ligase activity and coordination of the CDK2-BRCA1-NPM pathway.
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Materials and Methods
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Antibodies, Expression Constructs and Purified Proteins. Mouse monoclonal antibodies to hemagglutinin (12CA5; Roche, Indianapolis, IN), Myc (9E10; BabCo, Richmond, CA), FLAG (M2; Sigma, St. Louis, MO), and 
-tubulin and ß-tubulin (DM1A+DM1B; Neomarkers, Fremont, CA) as well as rabbit polyclonal antibodies to BRCA1 (sc-642; Santa Cruz Biotechnology, Santa Cruz, CA) were purchased commercially. Rabbit polyclonal antibody to CDK2, cyclin E1, and cyclin B1 were previously described (9, 10). Rabbit polyclonal antibody to BARD1 for immunoblotting and full-length Flag-BRCA1 plasmid are generous gifts from Dr. Richard Baer (Pathology, Columbia University, New York, NY). Mammalian expression plasmids for BRCA1, BARD1, NPM (B23.1), HA-ubiquitin, HA-CDK1, CDK2, cyclinA1, B1, and E1 were previously described (2, 4, 6, 9, 11) . The point mutations were produced by site-directed mutagenesis (Stratagene, La Jolla, CA) according to the manufacturer's instructions. All plasmids used were verified by DNA sequencing. Recombinant NH2-terminal fragments of His-BARD1, 14-189 and 1-320 were purified as described (2).
Cell Culture, Transfection and Cell Synchronization. Cells were cultured in DMEM supplemented with 10% FCS and 1% antibiotic-antimycotic agent (Invitrogen, Carlsbad, CA). Cells were transfected using the standard calcium phosphate precipitation method. Total plasmid DNA (15 µg per 100 mm dish) was adjusted to equal amounts by adding the parental pcDNA3 vector. Cell synchronization by a double thymidine block was previously described (12). For the thymidine-nocodazole block, asynchronously growing HeLa cells were cultured in the presence of 2mmolthymidine for 18 hours, grown in medium without thymidine for 3hours, and then incubated with 100 ng/mL nocodazole for 12 hours. Cells were then released into fresh medium and harvested at indicated times. Cell cycle progression of propidium iodide–stained cells was monitored by flow cytometry analysis using FACSCalibur (Becton Dickinson, San Jose, CA).
Immunologic and Biochemical Techniques. Immunoprecipitation and immunoblotting methods, including the detection of in vivo ubiquitinated substrates, were previously described (4, 6). For dephosphorylation of immunoprecipitated HA-BARD1, the proteins immobilized to protein A beads were incubated with 2 units of calf intestinal alkaline phosphatase (Takara, Shiga, Japan) at 37°C for 30 minutes. For pulse-chase analysis, 293T cells were pulse-labeled with [35S]-methionine for 1hour and chased for the indicated lengths of time. Immunoprecipitates from the cells were resolved by SDS-PAGE as previously described (12). In vitro kinase assays were done as described (10, 11) using anti-CDK2 or anti–hemagglutinin immunocomplexes precipitated from 293T cells transfected with CDK2-cyclin E1 or HA-CDK1-cyclin B1, respectively, and 2 µg of substrate protein. To prepare nuclear and cytoplasmic protein fractions, cells were lysed by incubation at 4°C for 5 minutes with buffer A [10 mmol/L Tris-HCl (pH 7.4), 100 mmol/L NaCl, 0.5% Triton X-100, 2.5 mmol/L MgCl2, 10 mmol/L NaF, and proteinase inhibitors] and passed through a 26-gauge needle thrice. The cytoplasmic supernatant fraction was obtained after centrifugationat 4,000 x g for 2 minutes. The remaining pellet was extracted by sonication in buffer B [50 mmol/L Tris-HCl (pH 7.5), 150 mmol/L NaCl, 0.5% NP40, 50 mmol/L NaF, 1 mmol/L DTT, 1 mmol/L NaVO3, and proteinase inhibitors], yielding the nuclear fraction.
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Results
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Cell Cycle-Dependent Expression of BARD1. Through immunoblotting, we noticed that endogenous and transfected BARD1 migrated as two bands, a characteristic consistent with phosphorylated proteins. To determine the significance of the doublets, we first tested whether the migration pattern altered during the cell cycle. HeLa cells were arrested either at the G1-S boundary by a double thymidine block (Fig. 1A)) or in mitosis by a thymidine-nocodazole block (Fig. 1B) and then released to synchronously progress through the cell cycle. The expression level of BARD1 was highest during mitosis and seemed as a slower migrating band (Fig. 1A, lane 8, and B, lanes 1 to 3). This coincided with the highest expression of BRCA1 and of cyclin B1. At the M/G1 transition, BARD1 became a doublet (Fig. 1A, lane 9, and B, lane 4) and then switched to a predominantly faster migrating band when the cells entered G1 phase (Fig. 1A, lane 10, and B, lane5). With this came the disappearance of BRCA1 and cyclin B1proteins. The expression level of BARD1 was down-regulated during G1 and S phases, especially from late G1 phase when the predominantly faster migrating band became a faint doublet (Fig.1A, lane 2, and B. lane 8) and when cyclin E1 was expressed. The altered molecular weight and the steady state level of BARD1, accompanied by the expression of cyclins suggests that BARD1 could be a target of CDKs.
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Figure 1. Cell cycle expression of BARD1. HeLa cells were arrested by a double thymidine block (A) or a thymidine-nocodazole block (B), and progression through the cell cycle after release from the blocks was monitored by FACS analysis (bottom). Cells were analyzed by immunoblotting with the antibodies at the indicated times after release (top). Asyn, asynchronous cells.
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BARD1 is Phosphorylated by CDK1/2 on its NH2 Terminus. To determine if BARD1 is phosphorylated by CDKs, we tested whether coexpression of CDK/cyclins could cause the molecular weight shift of BARD1. Truncated fragments of HA-BARD1 were co-transfected into 293T cells with various CDK/cyclins, and HA-BARD1 fragments were immunoblotted. The mobility of HA-BARD1 NH2-terminal fragment 1-320 (HA-BARD11-320) was retarded by the coexpression of either of CDK/cyclin, whereas the mobility of COOH-terminal fragment 411-777 (HA-BARD1411-777) was unchanged (Fig. 2A). There were at least three slower migrating products detected after immunoblotting HA-BARD11-320 (Fig. 2A, B, and D, arrows) which collapsed upon alkaline phosphatase treatment (Fig. 2B) suggesting that they are phosphorylated forms of BARD1.
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Figure 2. CDK2-cyclin A1/E1 and CDK1-cyclin B1 phosphorylate BARD1 on its NH2 terminus. A, B and D, Myc-BRCA11-772 and either the NH2-terminal (1-320) or the COOH-terminal (411-777) fragment of HA-tagged BARD1 was cotransfected in 293T cells with CDK-cyclin or parental pcDNA3 vector. HA-BARD1 was immunoprecipitated and immunoblotted with anti-HA antibody. B, HA-BARD11-320 immobilized to agarose beads was incubated with alkaline phosphatase (AP+) or buffer alone (–). C, purified recombinant GST (lanes 1, 4, 7), His-BARD114-189 (lanes 2, 5, 8), and His-BARD11-320 (lanes 3, 6, 9) were resolved by SDS-PAGE and stained with Coomassie brilliant blue (CBB, left), or were incubated with [ -32P] ATP and immunocomplexes containing CDK2-cyclin E1 (lanes 4-6) or CDK1-cyclin B1 (lanes 7-9). Proteins were resolved by SDS-PAGE and autoradiographed. D, HA-BARD11-320 with indicated mutations were transfected instead of wild type. *, IgG. K2/E1, CDK2-cyclin E1; K2/A1, CDK2-cyclin A1; K1/B1, CDK1-cyclin B1. HA-BARD1-P indicates phosphorylated HA-BARD1.
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To test whether the kinases directly phosphorylate BARD1, we next examined purified recombinant NH2-terminal fragments of BARD1 for their capacity to be CDK substrates in vitro. Both His-BARD114-189 and His-BARD11-320 were phosphorylated by CDK2-cyclin E1 and CDK1-cyclin B1 in vitro (Fig. 2C). Consistent with the mobility shift of HA-BARD11-320 in vivo, the in vitro phosphorylated His-BARD11-320 also exhibited several bands, suggesting that more than one phosphorylation site exists in the fragment. The ability to in vitro phosphorylate bacterially purified BARD1 fragments suggests that BRCA1 is unnecessary for the kinase to interact. Supporting this idea, coexpression of BRCA1 does not affect the quantity of BARD1 phosphorylation in vivo (data not shown). We mapped four phosphorylation sites in BARD1 by mutational analyses and identified a quadruple mutant, BARD1 S148A/S251A/S288A/T299A, which exhibited no detectable mobility shift by either CDK2-cyclin E1 (Fig. 2D) or CDK1-cyclin B1 (data not shown). These results suggest that BARD1 is phosphorylated by CDK1 and CDK2 on its NH2 terminus in a manner independent of BRCA1-BARD1 heterodimer formation.
CDK2 Inhibits the Ubiquitin Ligase Activity of BRCA1-BARD1. We next investigated the possibility that CDK2 may affect NPM ubiquitination by the BRCA1-BARD1 ligase because NPM is regulated by CDK2-cyclin E1 phosphorylation during centrosome duplication (8, 13). Surprisingly, CDK2-cyclin E1/A1 but not CDK1-cyclin B1 completely abolished the ubiquitination of NPM by BRCA1-BARD1 in vivo (Fig. 3A). The inhibition of NPM ubiquitination by CDK2 is not due to NPM phosphorylation because NPMT199A, a mutant that abolishes the CDK2 phosphorylation site (13), remained a viable substrate of the BRCA1-BARD1 ligase that was still sensitive to CDK2 inhibition (Fig.3B). We then examined the effect of CDK2-cyclin E1/A1 on the intrinsic ligase activity of the BRCA1-BARD1 heterodimeric complex by measuring BRCA1 autoubiquitination. CDK2-cyclin E1/A1 completely abolished autoubiquitination of BRCA1, whereas CDK1-cyclin B1 did not (Fig. 3C). Because BARD1 is a substrate of CDK2-cyclin E1, inhibition of BRCA1-BARD1 ubiquitin ligase activity could be a direct effect of BARD1 phosphorylation. However, autoubiquitination of BRCA1 mediated by the unphosphorylatable BARD1 mutant, S148A/S251A/S288A/T299A, is still inhibited by CDK2-cyclin E1 (Fig. 3D). This suggests that the inhibition of ubiquitin ligase activity is not due to BARD1 phosphorylation.
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Figure 3. Cdk2-cyclin E1 and CDK2-cyclin A1 inhibit ubiquitin ligase activity of BRCA1-BARD1. A-C, 293T cells transfected with plasmids were boiled in 1% SDS lysis buffer, diluted to 0.1% SDS, and immunoprecipitated with anti-FLAG antibody (A and B) or anti-Myc antibody (C) followed by immunoblot with anti-HA antibody to detect hemagglutinin ubiquitin–conjugated proteins. D, in vivo autoubiquitinated BRCA11-222 was detected as in C with or without cotransfection of increasing amounts of CDK2-cyclin E1 (lanes 2 and 6, 0.2 µg; lanes 3 and 7, 0.6 µg; lanes 4 and 8, 2 µg each). MT, BARD1S148A/S251A/S288A/T299A; *, IgG.
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Expression of BRCA1 and BARD1 are Reduced by CDK2-Cyclin E1. We next examined whether CDK2 affects the protein expression level of BRCA1 and BARD1. Consistent with ubiquitin ligase activity, the steady state level of either BRCA1 or BARD1 was dramatically reduced by CDK2-cyclin A1/E1 (Fig. 4A, lanes 3 and 4) in a dose-dependent manner (Fig. 4B), but not by CDK1-cyclin B1 (Fig. 4A, lane 5). The ineffectiveness of CDK1-cyclinB1 to cause BRCA1-BARD1 instability is not due to failure of protein expression or of kinase activity in cells because phosphorylation of BARD1 was clearly detected (Fig. 4A, lane 5). Cells were then pulse-chased with [35S]-methionine. The half-life oftheBRCA1-BARD1 complex in cells is remarkably reduced by coexpression of CDK2-cyclin E1 (from 4-6 hours to < 2 hours; Fig. 4C), suggesting that the reduced expression is due to protein degradation.
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Figure 4. BRCA1 and BARD1 are exported to the cytoplasm and repressed by CDK2-cyclin E1 coexpression. A, 293T cells were transfected with indicated plasmids, immunoblotted with anti-HA antibody (top), and then reprobed with anti-Myc antibody (bottom). B, 293T cells in a 60 mm dish were transfected with plasmids encoding Myc-BRCA11-772, HA-BARD1 (lanes 1-4, 1 µg each) and increasing amounts of CDK2 and cyclin E1 (lanes 2, 3 and 4, 0.1, 0.5, and 1.5 µg each). The steady state level of each protein was analyzed by immunoblot using the indicated antibodies (top, anti-HA; second portion, anti-HA followed by anti-Myc reprobe). C, 293T cells transfected with Myc-BRCA11-772, HA-BARD1, and parental pcDNA3 vector (top) or CDK2-cyclin E1 (bottom) were pulsed with [35S]-methionine for one hour and chased for the indicated lengths of time. Cell lysates were immunoprecipitated with anti-Myc antibody, resolved by SDS-PAGE and autoradiographed. D, 293T cells were transfected with FLAG-BRCA1, HA-BARD1, and parental pcDNA3 vector (lanes 1 and 2) or CDK2-cyclin E1 (lanes 3 and 4). MG132 (20 µmol/L) was added to the cells 6 hours before harvesting. Cells were fractionated into nuclear (N) and cytoplasmic (C) pools, followed by immunoblot with the indicated antibodies. The second portion presents a longer exposure.
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BRCA1 and BARD1 are Exported to the Cytoplasm by CDK2-Cyclin E1. Because it was reported that nuclear export of BRCA1 and BARD1 to the cytoplasm causes their degradation (14), we tested whether coexpression of CDK2 and cyclin E1 affects the localization of BRCA1. BRCA1 and BARD1 were transiently transfected into 293T cells with or without CDK2 and cyclin E1. The proteasome inhibitor carbobenzoxyl-leucinyl-leucinyl-leucinal (MG132) was added to the cells to inhibit the degradation of cytoplasmic BRCA1. To quantify BRCA1 protein levels in the nucleus and cytoplasm, we fractionated transfected cells and analyzed each fraction by immunoblot analysis. In the absence ofCDK2 and cyclin E1, the BRCA1 protein level in the cytoplasm was lower than that observed in the nucleus (Fig. 4D, top,lanes 1 and 2). Upon coexpression of CDK2 and cyclin E1, the level of cytoplasmic BRCA1 protein exceeded the level observed in the nucleus (middle, lanes 3 and 4). These results suggest that CDK2-cyclin E1 coexpression causes BRCA1 nuclear export.
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Discussion
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Here we report that the BRCA1-BARD1 ubiquitin ligase is down-regulated by CDK2-cyclin E1/A1 but not by CDK1-cyclin B1. Although the NH2 terminus of BARD1 is directly phosphorylated by CDK2, the phosphorylation itself is not likely important for the down-regulation because an unphosphorylatable mutant of BARD1 remained sensitive to ubiquitin ligase inhibition by CDK2, and although CDK1 phosphorylates BARD1 at the same site as CDK2, it did not inhibit BRCA1-BARD1 ligase activity. Upon coexpression of CDK2-cyclin E1, the BRCA1-BARD1 complex is degraded and, simultaneously, BRCA1 is exported from the nucleus to the cytoplasm. It remains to be determined whether the degradation results from the loss of nontraditional ubiquitin ligase activity or whether the degradation upon nuclear export causes the loss of the activity.
One discrepancy between our data and other published data is the observation that BRCA1 and BARD1 are sequestered within discrete nuclear domains during the S and G2 phases of the cell cycle while CDK2-cyclin E1/A1 are active (15, 16). The subnuclear translocation of BRCA1 and BARD1 upon DNA damage suggests that their role is played during S and G2 phases (15). In seeming contrast to this data, we and others showed by Western analysis that the level of BRCA1 protein is highest in G2-M phases (16, 17 , and Fig. 1). Intriguingly, a recent report showed that the level of proteasome-sensitive ubiquitin conjugates of BRCA1 is highest during S phase, implying greater turnover of BRCA1 in S phase cells (17). We hypothesize that the increase in BRCA1 turnover could be regulated in a CDK2-cyclin E1/A1–dependent manner. Furthermore, the same report showed that BARD1 expression also peaks during the G2-M phases (17), which is consistent with our observation (Fig. 1). An interesting question that remains to be investigated is the mechanism by which the BRCA1 and BARD1 complexes residing within the nuclear dots escape degradation during S phase.
We recently showed that the BRCA1-BARD1 ligase ubiquitinates and stabilizes NPM (6). Coincidentally, NPM is also phosphorylated by CDK2-cyclin E1. The phosphorylation of NPM by CDK2-cyclin E1 at the G1-S transition causes dissociation of NPM from the centrosome and allows it to initiate the duplication process. NPM remains dissociated from the centrosome during Sphase, while daughter centrioles develop into two mature centrosomes (spindle poles; refs. (8, 13). Together with the results presented here, CDK2 may affect NPM in two mutually exclusive ways, by direct phosphorylation to control NPM association with the centrosome, and by protecting NPM from BRCA1-BARD1 ubiquitination to promote its instability. Hence, theCDK2-BRCA1-NPM pathway may coordinately regulate centrosome duplication.
Finally, our results may shed some light on the mechanism by which some sporadic breast cancers are sustained. A poor prognosis is significantly correlated with tumor tissues that express higher cyclin E1 protein levels (18). According to our data, the higher cyclin E1 levels could dampen BRCA1 ubiquitin ligase activity. Future work exploring the detailed mechanism of the CDK2-BRCA1-NPM pathway may provide a role for BRCA1 in sporadic breast cancer.
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Acknowledgments
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Grant support: Grants from the Japan Society for the Promotion of Science, and the Japanese Ministry of Education, Culture, Sports, Science and Technology.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
We thank Drs. Rachel E. Klevit and Masatoshi Kitagawa for helpful discussions and critical reading of the manuscript, and Takako Kuwahara for secretarial assistance.
Received 7/15/04.
Revised 10/13/04.
Accepted 11/ 5/04.
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Top
Abstract
Introduction
