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DNA Damage–Induced BARD1 Phosphorylation Is Critical for the Inhibition of Messenger RNA Processing by BRCA1/BARD1 Complex

¹ Genetics of Development and Disease Branch, National Institute of Diabetes and Digestive and Kidney Diseases, NIH, Bethesda, Maryland and ² Chemistry Department, Hunter College, City University of New York, New York, New York

Requests for reprints: Sean Bong Lee, Genetics of Development and Disease Branch, National Institute of Diabetes and Digestive and Kidney Diseases, NIH, 9000 Rockville Pike, Bethesda, MD 20892. Phone: 301-496-9739; Fax: 301-480-0638; E-mail: seanL{at}intra.niddk.nih.gov.

	Abstract

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BRCA1-associated RING domain protein BARD1, along with its heterodimeric partner BRCA1, plays important roles in cellular response to DNA damage. Immediate cellular response to genotoxic stress is mediated by a family of phosphoinositide 3-kinase–related protein kinases, such as ataxia-telangiectasia mutated (ATM), ATM and Rad3-related, and DNA-dependent protein kinase. ATM-mediated phosphorylation of BRCA1 enhances the DNA damage checkpoint functions of BRCA1, but how BARD1 is regulated during DNA damage signaling has not been examined. Here, we report that BARD1 undergoes phosphorylation upon ionizing radiation or UV radiation and identify Thr⁷¹⁴ as the in vivo BARD1 phosphorylation site. Importantly, DNA damage functions of BARD1 (i.e., inhibition of pre-mRNA polyadenylation and degradation of RNA polymerase II) are abrogated in T714A and T734A mutants. Our findings suggest that phosphorylation of BARD1 is critical for the DNA damage functions of the BRCA1/BARD1 complex. (Cancer Res 2006; 66(9): 4561-5)

	Introduction

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Protein phosphorylation is critical in the cellular response to DNA damage, acting as a molecular switch that regulates many important DNA damage checkpoint responses. The principal kinases involved in this signaling process are members of the phosphoinositide 3-kinase–related protein kinase (PIKK) family, such as ataxia-telangiectasia mutated (ATM) and ATM and Rad3-related (ATR; refs. 1, 2). Many of the ATM/ATR kinase substrates, such as BRCA1, CHK2, NBS1, MRE11, p53, and SMC1, signal cell cycle checkpoint responses to DNA damage, playing important roles in cell cycle arrest, apoptosis, and DNA repair (3). The tumor suppressor BRCA1 is phosphorylated by ATM upon DNA damage, and together with BARD1, performs multiple functions in the DNA damage responses, including in DNA repair, in transcription, and in RNA processing (4, 5). BRCA1/BARD1 form a complex through their respective NH₂-terminal RING domains and exhibit significant E3 ubiquitin ligase activity (5). Through its ubiquitin ligase activity, the BRCA1/BARD1 complex can undergo autoubiquitination (6, 7) and can ubiquitinate substrates, such as p53 (8), Nucleophosmin/B2 (9), -tubulin (10), and RNA polymerase II (RNAP II; refs. 11, 12). BRCA1/BARD1–mediated ubiquitination of RNAP II targets it for proteasome-mediated degradation and subsequent inhibition of transcription and RNA processing in response to genotoxic stress (11). In contrast to BRCA1, whose function is regulated by phosphorylation in response to genotoxic stress, how BARD1 activity is regulated and whether BARD1 is phosphorylated during DNA damage have not been examined.

	Materials and Methods

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Cell culture, antibodies, and constructs. Ataxia-telangiectasia fibroblasts (GM02052D and GM03487E) and control human fibroblasts (GM07532A and GM08398) were obtained from the Coriell Cell Repository (Camden, NJ). Rabbit polyclonal anti-BARD1 and monoclonal anti-CstF64 antibodies were kindly provided by Richard Baer (Columbia University) and James Manley (Columbia University), respectively. NH₂-terminal FLAG epitope-tagged human BARD1 cDNA was amplified by PCR and cloned into pCMV-Sport6. Quick Site–directed mutagenesis kit (Stratagene, La Jolla, CA) was used to generate Ala substitutions at indicated sites. All constructs were verified by sequencing.

In vivo labeling of ³²P-orthosphosphate. U2OS cells stably transfected with different BARD1 mutant constructs were starved for 30 minutes with phosphate-free DMEM, and one set of plates was treated with 10 Gy ionizing radiation. Phosphate-free DMEM containing 200 µCi/mL of ³²P-orthophosphate was added immediately, and cells were incubated for 2.5 hours. Cells were lysed and immunoprecipitated using anti-FLAG antibody M2 (Sigma, St. Louis, MO) and Protein G-Sepharose (Amersham, Arlington Heights, IL). Samples were resolved by SDS-PAGE and transferred to nitrocellulose membrane followed by autoradiography. The membrane was subsequently immunoblotted with anti-FLAG M2 antibody.

Generation of phospho-specific p-T714 BARD1 antibody. Phospho-peptide p-T714 [CKPKPDSDVT(PO₃)QTINTVA] was synthesized, conjugated to keyhole limpet hemocyanin (KLH), and used to immunize rabbits (Bethyl Laboratories, Montgomery, TX). The same phospho-peptide was used for the affinity purification of phospho-specific antibody.

	Results

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To determine whether BARD1 is modified following DNA damage, U2OS cells were irradiated with ionizing radiation, and BARD1 was examined by immunoblotting. As early as 30 minutes after -irradiation (10 Gy), appearance of a slower migrating form of BARD1 in SDS-PAGE was observed (Fig. 1A ). Treatment of HeLa or U2OS cells with doxorubicin, a chemotherapeutic agent that causes double-stranded DNA breaks, also led to the slower migration of BARD1 protein (Fig. 1B). Interestingly, only the BARD1 in the nuclear fraction was modified following DNA damage (Fig. 1B). To examine whether single-strand DNA damage can also result in BARD1 modification, U2OS cells were exposed to UV radiation, and the mobility of BARD1 was examined. As shown with ionizing radiation and doxorubicin treatments, the slow migrating form of BARD1 can be detected shortly after UV treatment (Fig. 1C).

View larger version (14K): [in this window] [in a new window]   Figure 1. BARD1 phosphorylation in response to DNA damage. A, U2OS cells were -irradiated at 10 Gy, and nuclear extracts were prepared at indicated times and immunoblotted with anti-human BARD1 antibody. B, nuclear and cytoplasmic extracts from either U2OS or HeLa cells, treated with either 1 or 5 µg/mL doxorubicin (Dox), were prepared and immunoblotted with anti-BARD1 antibody. C, U2OS cells were either treated with 1 µg/mL doxorubicin or with UV (100 J/m2), and nuclear extracts were prepared at indicated times were immunoblotted with anti-BARD1 antibody. D, nuclear extracts from U2OS cells, either untreated or treated with 1 µg/mL doxorubicin, were incubated with either tyrosine phosphatase (Y) or phosphatase (; New England Biolabs, Ipswich, MA) for 30 minutes and immunoblotted with anti-BARD1 antibody. IR, ionization radiation.

Because a family of PIKKs, such as ATM and ATR kinases, plays important roles in the DNA damage response (1, 2), we sought to test whether PIKK is involved in BARD1 phosphorylation upon DNA damage. Incubation of U2OS or HeLa cells with increasing amounts of caffeine, an inhibitor of PIKKs, effectively abolished BARD1 phosphorylation induced by doxorubicin, suggesting that phosphorylation of BARD1 is PIKK dependent (Fig. 2A ). To test directly whether ATM is responsible for BARD1 phosphorylation, two independently derived primary human fibroblast cell lines from ataxia-telangiectasia patients were treated with either doxorubicin or ionizing radiation along with two control primary cell lines. In both ataxia-telangiectasia cells, BARD1 phosphorylation is reduced in response to either treatment compared with controls (Fig. 2B), showing that ATM is at least partly responsible for BARD1 phosphorylation. Residual phosphorylation seen in the ataxia-telangiectasia cells, however, indicates that other kinases can also phosphorylate BARD1.

View larger version (15K): [in this window] [in a new window]   Figure 2. BARD1 phosphorylation is mediated by ATM and requires BRCA1. A, nuclear extracts prepared from U2OS or HeLa cells treated with 1 µg/mL doxorubicin (Dox) in the absence or presence of 2 or 5 mmol/L caffeine were immunoblotted with anti-BARD1 antibody. B, nuclear extracts prepared from normal human fibroblast cells (N1 and N2) or A-T cells (A-T1 and A-T2) treated with or without 1 µg/mL doxorubicin or -irradiated at 10 Gy (ionization radiation, IR) were immunoblotted with anti-BARD1 antibody. Nuclear extracts from U2OS were used as a control. C, U2OS or HCC1937 (BRCA1 mutant) cells were treated with either 1 µg/mL doxorubicin for 16 hours or ionization radiation (10 Gy). Nuclear extracts were prepared at indicated times and immunoblotted with anti-BARD1 antibody. D, HCT15 cells were treated with either 1 µg/mL doxorubicin or 10 Gy ionization radiation. Nuclear extracts were prepared and immunoblotted with anti-BARD1 or anti-phospho-p53 (Ser15) antibodies (Cell Signaling, Danvers, MA).

CHK2 is an effector kinase downstream of ATM, which can also phosphorylate ATM-substrates, such as BRCA1 and p53, in response to DNA damage (3). To determine whether BARD1 phosphorylation is also mediated by CHK2 kinase, we examined BARD1 phosphorylation in human colon cancer HCT15 cell line, which has extremely low levels of endogenous CHK2 kinase activity due to compound mutations in CHEK2 that lead to unstable proteins (14). In HCT15 cells treated with either doxorubicin or ionizing radiation, BARD1 phosphorylation was readily observed (Fig. 2D). As expected, we observed the Ser¹⁵ phosphorylation of p53 by ATM. These results suggest that CHK2 is dispensable for the observed DNA damage–induced phosphorylation of BARD1.

ATM/ATR kinases phosphorylate serine or threonine residues, which are immediately followed by a glutamine residue (SQ/TQ; ref. 3). Examination of primary sequence revealed that there are four potential ATM/ATR phosphorylation sites (SQ/TQ) in the human BARD1 (Thr¹⁶⁵, Ser²⁴⁴, Thr⁷¹⁴, and Thr⁷³⁴). Sequence comparison with other BARD1 orthologues revealed that the last two TQ motifs located in the second BRCT domain (Thr⁷¹⁴ and Thr⁷³⁴) are evolutionarily conserved (Fig. 3A ). To determine which of these ATM/ATR phosphorylation sites are modified in response to genotoxic stress, simultaneous or individual mutations of the four Thr/Ser to Ala were analyzed. When cells were transfected with the BARD1 mutant that contains AQ substitutions at all four SQ/TQ sites (Quad), the BARD1 mutant was not phosphorylated after DNA damage (Supplementary Fig. S1A). To define the phosphorylation sites more precisely, U2OS cells were transfected with FLAG-tagged BARD1 expression vectors containing individual substitutions at each phosphorylation site and labeled with ³²P-orthophosphate after ionizing radiation treatment. Transient overexpression of the different BARD1 mutants in cells led to in vivo phosphorylation of the wild type (WT), T165A, S244A, and T714A versions of BARD1 but not in T734A and Quad mutants (Supplementary Fig. S1B). However, the observed phosphorylation was irrespective of DNA damage, prompting us to examine BARD1 phosphorylation under a physiologic condition. Thus, we next examined DNA damage–induced phosphorylation of BARD1 in cells stably expressing BARD1 mutants. As expected, WT BARD1 showed enhanced phosphorylation in response to ionizing radiation, but T714A, T734A, and Quad BARD1 mutants were not efficiently phosphorylated in response to DNA damage (Fig. 3B). This result suggests that Thr⁷¹⁴ and Thr⁷³⁴ may be important DNA damage phosphorylation sites. We also note that BARD1 is a phospho-protein in the absence of DNA damage.

View larger version (20K): [in this window] [in a new window]   Figure 3. BARD1 Thr714 and Thr734 are evolutionarily conserved and phosphorylated in vivo. A, amino acid sequence alignment near the COOH-terminal BRCT domain of different BARD1 orthologues. Boxes indicate conserved PIKK phosphorylation (TQ) sites. T714 and T734 refer to the human BARD1 residues. B, U2OS cells stably transfected with different BARD1 expression constructs were either untreated or ionization radiation treated (10 Gy) and then metabolically labeled with 32P-orthophosphate (200 µCi/mL). Cell extracts were immunoprecipitated with anti-FLAG antibody M2 (Sigma), resolved by SDS-PAGE, and transferred to nitrocellulose membrane followed by autoradiography (P32-IP). Subsequently, membrane was immunoblotted with anti-FLAG M2 antibody (-FLAG). C, nuclear extracts from U2OS cells treated with 1 µg/mL doxorubicin (left) or increasing amounts of doxorubicin (1, 5, or 10 µg/mL; right) were immunoblotted with phospho-specific p-T714 BARD1 antibody. Where indicated, nuclear extracts were treated with phosphatase for 30 minutes before immunoblotting (top left). The same membrane was stripped and blotted with anti-BARD1 antibody (bottom left).

Recent reports have shown that in response to DNA damage BRCA1/BARD1 complex ubiquitinates RNAP II (11, 12) and subsequently leads to a rapid degradation of RNAP II by the proteasome. Thus, we next examined whether the DNA damage–induced BARD1 phosphorylation is important for the degradation of RNAP II after DNA damage. Consistent with previous results, UV treatment reduced the accumulation of both hypophosphorylated (RNAP IIA) and hyperphosphorylated (RNAP IIO) forms of RNAP II in cells stably transfected with empty vector or WT BARD1 (Fig. 4A ). In contrast, cells stably transformed with the T714A or the T734A versions of BARD1 showed a stabilization of both RNAP II isoforms following UV treatment, especially of the RNAP IIO isoform. This is consistent with a previous observation that RNAP IIO is the target of BRCA1/BARD1 ubiquitination (11, 12). This result suggests that phosphorylation of BARD1 at Thr⁷¹⁴ and Thr⁷³⁴ is important for the preferential degradation of RNAP IIO mediated by the BRCA1/BARD1 complex in response to DNA damage.

View larger version (24K): [in this window] [in a new window]   Figure 4. BARD1 T714A and T734A mutants are defective in RNAP II degradation and mRNA cleavage inhibitory activity in response to DNA damage. A, U2OS cells stably transformed with various BARD1 mutants were untreated or treated with UV (20 J/m2). After 2 hours, cell extracts were immunoblotted with indicated antibodies: BRCA1 (C-20, Santa Cruz Biotechnology, Santa Cruz, CA), RNAP II (8WG16, Covance, Berkeley, CA), and RNAP IIO (H5, Covance, Berkeley, CA). Anti-actin (A2066, Sigma) blot shows relatively equal loading in all samples. B, nuclear extracts were prepared from cells stably transformed with various BARD1 mutants, either untreated or treated with UV (20/m2). In vitro RNA cleavage assay was done as previously described (15) using SV40 late precursor RNA (SVL). 5' cleaved product and SVL precursor RNA are denoted. C, nuclear extracts prepared from various stable BARD1 expressing cells, either untreated or treated with UV (20 J/m2), were immunoprecipitated with anti-CstF64 antibody. Supernatants and the immunoprecipitated pellets were resolved by SDS-PAGE and immunoblotted with anti-FLAG M2, anti-CstF64, or anti-actin antibodies.

The transient inhibition of 3' RNA processing following DNA damage reflects the formation of the BRCA1/BARD1/CstF complex (15). To test the effect of BARD1 phosphorylation on the BRCA1/BARD1/CstF complex formation, we analyzed the complex in nuclear extracts from UV-treated cells expressing different mutants of BARD1. As the BARD1/CstF-50 interaction involves the intact CstF complex (15), we used monoclonal antibodies against CstF-64, another CstF subunit, to immunoprecipitate the complex. As shown in Fig. 4C, T734A BARD1 mutant did not form a complex with CstF, irrespective of UV treatment, whereas WT BARD1 was able to form a complex that increased significantly after the UV damage. Unexpectedly, T714A version of BARD1 still formed a complex with CstF even in the absence of genotoxic stress; however, unlike the WT BARD1, this interaction did not increase with DNA damage (Fig. 4C). The results indicate that the phosphorylation of BARD1 plays an important role in the BARD1/CstF interaction and subsequent inhibition of CstF activity. In contrast, BARD1/BRCA1 interaction was still retained in the T714A, T734A, and Quad BARD1 mutants (Supplementary Fig. S2).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
DNA damage leads to different cellular responses, such as cell cycle arrest, inhibition of transcription and of RNA processing, DNA repair, and apoptosis. ATM/ATR kinases are able to control many aspects of the DNA damage response by phosphorylating specific substrates important in different cellular pathways (2, 3). Of the four potential ATM/ATR phosphorylation sites in human BARD1, only two TQ sites near the tandem BRCT motifs are evolutionarily conserved (Fig. 3A), suggesting the importance of BARD1 phosphorylation at these residues. Our study shows that BARD1 is phosphorylated at Thr⁷¹⁴, which increased with DNA damage, using phospho-specific p-T714 antibody (Fig. 3C). Our results also revealed that BARD1 exists as a phospho-protein, which is hyperphosphorylated in response to DNA damage.

Mutation of either T714A or T734A significantly reduced DNA damage–induced phosphorylation of BARD1 (Fig. 3B) and further resulted in a dysfunctional BARD1 in mediating inhibition of 3' RNA processing and degradation of RNAP II after DNA damage (Fig. 4). Loss of UV-induced inhibition of RNA processing in T734A mutant might be due to its inability to form a complex with CstF (Fig. 4C), suggesting that phosphorylation of Thr⁷³⁴ may be important for the DNA damage–induced BARD1/CstF interaction. Surprisingly, unlike the T734A substitution, T714A mutation did not abolish BARD1/CstF interaction (Fig. 4C). This observation suggests that the DNA damage–induced inhibition of 3' processing by BARD1 may not simply be due to sequestration of CstF but implicates a more direct role in the inhibition of CstF complex. Our study, thus, provides mechanistic insights by which BARD1 activity can be regulated by PIKK-mediated phosphorylation. Although the half-life of both T734A and Quad BARD1 mutants was reduced compared with WT or other BARD1 mutants (Supplementary Fig. S3A), it is unlikely that T734A and Quad mutants are grossly misfolded because these BARD1 mutants retained the ability to interact with BRCA1 (Supplementary Fig. S2). However, we cannot exclude the possibility that the loss of BARD1 function in T734A mutant may be due to local conformational changes in the BRCT domain because a truncated BARD1 containing only the NH₂-terminal RING domain can still interact with BRCA1 (5). Degradation of the mutant BARD1 was delayed with proteasome inhibitor MG132, suggesting that the observed instability of BARD1 was due to proteasome-mediated degradation (Supplementary Fig. S3A). These results suggest that phosphorylation of BARD1 may also be an important determinant of BARD1 stability.

It is likely that BARD1 has additional DNA damage–induced phosphorylation sites other than Thr⁷¹⁴ and Thr⁷³⁴, and that additional phosphorylation may also regulate different aspects of BARD1 function. Consistent with this view, recent reports showed that BARD1 can be phosphorylated by a cyclin-dependent kinase (CDK)/cyclin complex in a cell cycle–dependent manner (16, 17), and mutations in the CDK2/cyclin E1/A1 phosphorylation sites of BARD1 confer increased sensitivity to mitomycin C treatment (16). The precise mechanisms by which CDK/cyclin– or PIKK-mediated BARD1 phosphorylation regulate its activity is not known. One possibility is that the phosphorylation sites of BARD1 may directly be involved in the binding of other proteins (as in Thr⁷³⁴ phosphorylation, leading to formation of BARD1/CstF complex) or may indirectly influence protein-protein interaction by inducing conformational changes. Because the BRCT domain serves as a phospho-peptide binding module (18, 19), DNA damage–induced phosphorylation of Thr⁷¹⁴ and Thr⁷³⁴ residues of BARD1 (which are located in the second BRCT domain) may convert the BRCT domain from a phospho-peptide binding module into a phospho-protein docking site for other phosphorylation-specific binding proteins. Additionally, although not mutually exclusively, Thr⁷¹⁴ and Thr⁷³⁴ phosphorylation and CDK/cyclin–mediated phosphorylation may also serve to activate or enhance the activity of BARD1 complex, such as its E3 ubiquitin ligase activity or homology-directed DNA repair (20). Elucidation of BARD1 structure with interacting peptides or a phosphorylated BARD1 may provide further insight into the mechanisms of BARD1 regulation and action during cellular response to DNA damage.

	Acknowledgments

 
Grant support: National Institute of General Medical Sciences-Score grant S06 60654 (F.E. Kleiman) and Intramural Research Program of the NIH/National Institute of Diabetes and Digestive and Kidney Diseases (S.B. Lee).

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. Richard Baer and James Manley for kindly providing the BARD1 and CstF64 antibodies; Drs. Dan Haber (Massachusetts General Hospital, Charlestown, MA), Chuxia Deng (NIH, Bethesda, MD), and Michael Kastan (St. Jude, Memphis, TN) for reagents; Dr. Eric McIntush (Bethyl Laboratories) for his advice on generating the BARD1 phospho-specific antibodies; Barbara Christensen and Lale Evsen for technical assistance; William DeGraff for his help with -irradiation; Drs. Richard Baer, Rick Proia, and Chuxia Deng for helpful discussion and critical reading of the article; and the anonymous reviewers for improving our article.

	Footnotes

 
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

H-S. Kim, H. Li, and M. Cevher contributed equally to this study.

H-S. Kim is currently at the Department of Biochemistry, College of Medicine, The Catholic University of Korea, Seoul 137-701, Korea.

Competing interests statement: The authors declared no competing interests.

Received 10/11/05. Revised 3/ 6/06. Accepted 3/14/06.

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This Article Abstract Full Text (PDF) Supplementary Data Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kim, H.-S. Articles by Lee, S. B. Search for Related Content PubMed PubMed Citation Articles by Kim, H.-S. Articles by Lee, S. B.