The BRCA2 activation domain associates with and is ... - Nature

Oncogene (2000) 19, 4441 ± 4445 ã 2000 Macmillan Publishers Ltd All rights reserved 0950 ± 9232/00 $15.00 www.nature.com/onc

SHORT REPORT

The BRCA2 activation domain associates with and is phosphorylated by a cellular protein kinase Jonathan Milner1,3, FrancËois Fuks1,3, Luke Hughes-Davies1,2 and Tony Kouzarides*,1 1 2

Wellcome/CRC Institute and Department of Pathology, Cambridge University, Tennis Court Road, Cambridge, CB2 1QR, UK; CRC Human Cancer Genetics Research Group, Addenbrooke's Hospital, Cambridge, UK

A substantial proportion of familial breast cancers have mutations within the BRCA2 gene. The product of this gene has been implicated in DNA repair and in the regulation of transcription. We have previously identi®ed at the amino-terminus of BRCA2 a transcriptional activation domain whose importance is highlighted by the presence of predisposing mutations and in-frame deletions in breast cancer families. This activation domain shows sequence similarity to a region of c-Jun which has been de®ned as a binding site for the c-Jun Nterminal kinase. Here, we show that the analogous region in BRCA2 is also a binding site for a cellular kinase, although this kinase is distinct from JNK. The BRCA2 associated enzyme is able to phosphorylate residues within the BRCA2 activation domain. Consistent with this observation, we ®nd that the activation domain of BRCA2 is phosphorylated in vivo. Our results indicate that the BRCA2 activation domain possesses a binding site for a kinase that may regulate BRCA2 activity by phosphorylation. Oncogene (2000) 19, 4441 ± 4445. Keywords: BRCA2; protein kinase; phosphorylation

An estimated 5 ± 10% of all breast cancer cases result from a hereditary predisposition to the disease. Germline mutations in the human BRCA2 gene account for a substantial proportion of familial, early-onset breast cancers (Wooster et al., 1995; Tatvtigian et al., 1996). BRCA2 has exons and encodes a very large protein of 3418 amino-acids (Tatvigian et al., 1996). The functions of BRCA2 remain unclear but accumulating evidence suggest that it may have a role in DNA repair. BRCA2 nullizygous mouse embryos are highly sensitive to ionizing radiation (Sharan et al., 1997) and are defective in DNA repair (Suzuki et al., 1997; Connor et al., 1997; Patel et al., 1998). Moreover several groups have reported an interaction between BRCA2 and Rad51, a protein involved in homologous recombination and DNA double-strand break repair (Sharan et al., 1997; Mizuta et al., 1997; Wong et al., 1998). The sites of Rad51 interaction in BRCA2 have been ®ne-mapped to the eight evolutionarily conserved BRC motifs (Wong et al., 1998; Chen et al., 1998) and to a C-terminal region (Sharan et al., 1997; Mizuta et al., 1997).

*Correspondence: T Kouzarides 3 These authors contributed equally to this work Received 25 May 2000; revised 10 July 2000; accepted 10 July 2000

In addition to a role in DNA repair, several lines of evidence suggest that BRCA2 may function to regulate transcription. We have previously shown that the amino-terminal exon 3 of BRCA2 has the ability to stimulate transcription when fused to GAL4-DNA binding domain (Milner et al., 1997). The activation domain within exon 3 was found to be bipartite, with a primary activating region (residues 18 ± 60) and an auxiliary activating region (residues 60 ± 105). Importantly, the introduction of a missense mutation in exon 3 (mutation Y42C) which is detected in familial breast cancer disrupts the activation potential of BRCA2 (Milner et al., 1997). This ®nding suggest that elimination of BRCA2 transactivation capacity might be an important event in the generation of a subset of inherited breast cancers. This concept is reinforced by the recent identi®cation of a deletion disrupting the whole BRCA2 exon 3 as the disease-causing mutation in a breast cancer family (Nordling et al., 1998). It is worth mentioning that not only fragments of BRCA2 are able to regulate transcription but that also the intact protein is able to do so. Indeed, exogenous expression of full-length BRCA2 was found to regulate p53's transcriptional activity (Marmorstein et al., 1998). Further evidence that BRCA2 may have a role in transcriptional regulation comes from our recent observation that BRCA2 N-terminus interacts with a transcriptional co-activator protein, P/CAF, and displays associated acetyltransferase activity when bound to it (Fuks et al., 1998). We have previously recognized that the auxiliary activating region of exon 3 (residues 60 ± 105) shows sequence similarity to the transcription factor c-Jun (Milner et al., 1997). In particular, the homology spans the d-region of c-Jun activation domain which contains the binding site for the c-Jun kinase JNK (Hibi et al., 1993; DeÂrijard et al., 1994) (see Figure 1a). In the present study, we have further investigated this sequence similarity of exon 3 activation domain to the c-Jun d-region. We ®nd that BRCA2 exon 3 does not bind to JNK but instead binds a distinct kinase. We also show that this protein kinase phosphorylates BRCA2 and that the phosphorylation event requires the activation domain of BRCA2. These results suggest that the mechanism by which BRCA2 activity is regulated may be, at least partly, through its association with, and phosphorylation by protein kinase. The sequence homology we have previously noted between the activation domain of BRCA2 and the JNK-binding site in c-Jun prompted us to examine whether JNK is binding to BRCA2. Given that JNK can bind to c-Jun and phosphorylate it, we asked if

Phosphorylation of BRCA2 activation domain by a cellular kinase J Milner et al

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Figure 1 BRCA2 c-Jun homology region does not bind to JNK1. (a) BRCA2 exon 3 activation domain shows sequence similarity with the JNK-binding site in c-Jun (d-region). The sequence alignment is shown between BRCA2 activation domain from aminoacids 48 ± 105 and c-Jun activation domain from amino-acids 1 ± 55, which encompass the JNK-binding site (d-region). Residues with identity or similarity are shaded. Below the alignment is shown a schematic representation of BRCA2 with its amino-terminal exon 3 underlined. Exon 3 contains, as previously described (Milner et al., 1997), a bipartite activation domain with a primary activating region (PAR, residues 18 ± 60) and an auxiliary activating region (AAR, residues 60 ± 105). (b) A solid-phase kinase assay was used to test for binding of BRCA2 to JNK1. Recombinant JNK1 was incubated with equivalent amounts of bacterially expressed GST (lane 1) or GST BRCA2 60 ± 105 (containing the c-Jun homology region; lane 2) or, as a positive control, GST c-Jun 1 ± 79 (lane 3). After 1 h at 4 8C, the beads were washed in kinase buer (25 mM HEPES pH 7.4, 5 mM b-mercaptoethanol, 10 mM MgCl2, 3 mM Na3VO4). The beads were then incubated with 1 mCi for [g-32P]ATP in kinase buer for 15 min at 308C. The phosphorylated proteins were separated by SDS ± PAGE and visualized by autoradiography. Expression and puri®cation of GST fusions were done as previously reported (Fuks et al., 2000)

BRCA2 can bind JNK and become phosphorylated by it. To this end, we performed solide-phase kinase assays (Hibi et al., 1993; DeÂrijard et al., 1994), in which JNK was incubated with immobilized-bacterially expressed GST BRCA2, extensively washed and radiolabelled ATP added to test for phosphorylation of BRCA2. The N-terminal activation domain of c-Jun (residues 1 ± 79) linked to GST was used as a positive control. As expected, GST c-Jun 1 ± 79 was eciently phosphorylated by recombinant JNK1, indicating that there was an association between c-Jun and JNK1 (Figure 1b, lane 3). No phosphorylation of the control GST alone was observed (lane 1). In contrast to GST c-Jun, residues 60 ± 105 of BRCA2 (encompassing the sequence homology with the c-Jun d-domain) failed to associate with JNK1 kinase activity. Indeed, when we used the solide-phase kinase assay with immobilized Oncogene

GST BRCA2 60 ± 105 no phosphorylation by recombinant JNK1 was detected (Figure 1b, lane 2). Thus, these data indicate that JNK1 does not bind to BRCA2 activation domain and are in agreement with May et al. (1999). JNK1 belongs to the JNK family of protein kinases, which includes at least 10 isoforms, and which are all able to bind to and phosphorylate the c-Jun d-domain (Gupta et al., 1996). Thus, although BRCA2 was not binding to JNK1, it was still possible that another JNK family member, or even a distinct kinase with a similar recognition site to JNK, was binding to and phosphorylated the BRCA2 c-Jun homology domain. To address this possibility, we carried out solide-phase kinase assays as described above, this time with Hela nuclear extracts. Figure 2a (lane 1) shows that immobilized GST BRCA2 18 ± 141, which contains


the d-homology region, is eciently phosphorylated in vitro when incubated with Hela nuclear extracts. In contrast, no kinase activity was detected when GST alone was used (lane 3). These results indicate that a protein kinase present in Hela cells is speci®cally able to bind and phosphorylate in vitro BRCA2 exon 3 activation domain. To examine whether the BRCA2associated kinase activity was mediated through the dhomology domain (i.e. residues 60 ± 105), we used a mutated version of BRCA2 18 ± 141 in which the dhomology region (60 ± 105) was deleted (GST BRCA2 18 ± 141 Dd). As depicted in Figure 2 (lane 2), removal of this region markedly reduced the kinase activity associated to BRCA2 exon 3. We next wished to de®ne the kinase-binding-site in BRCA2. The above-described experiment using the mutant GST BRCA2 18 ± 141 Dd (Figure 2a) did not distinguish whether this mutant aected the phosphoacceptor sites or the binding site for the kinase. Thus, to directly determine whether the d-homology region of BRCA2 was binding to the kinase, we

Figure 2 A kinase from Hela cells binds and phosphorylates in vitro the BRCA2 activation domain. (a) Hela nuclear extracts were incubated with GST fusions of BRCA2 (lanes 1 and 2) or GST alone (lane 3). After 12 h of incubation followed by extensive washing in kinase buer, the solid-phase kinase assay was performed as described in Figure 1b. The phosphorylated proteins were resolved by SDS ± PAGE and visualized by autoradiography. (b) Elution of the cellular kinase from BRCA2 activation domain. Hela nuclear extracts were incubated with GST BRCA2 fusions or GST alone for 12 h. After extensive washing in kinase buer, GST fusion proteins bound to gluthatione-sepharose beads were subjected to elution in kinase buer containing 1 mM of cold ATP. After centrifugation, the eluted fraction was examined by phosphorylation of GST BRCA2 18 ± 141 in kinase buer containing 1 mCi of [g-32P]ATP for 15 min at 308C. Phosphorylation of BRCA2 18 ± 141 was analysed by SDS ± PAGE and autoradiography

examined whether the kinase eluted from GST BRCA2-loaded beads can phosphorylate exogenous BRCA2 18 ± 141 in solution. To this end, we performed a two step experiment in which we ®rst puri®ed the kinase from Hela nuclear extracts using GST BRCA2, then we attempted to elute the kinase from BRCA2 by the addition of cold ATP. Elution of the kinase was examined by phosphorylation in solution of GST BRCA2 18 ± 141 in the presence of radiolabelled ATP. Figure 2b indicates that the kinase binds speci®cally to the d-homology region of BRCA2. Indeed, the kinase is eciently eluted from GST BRCA2 18 ± 141 with cold ATP, as indicated by the ability of the kinase to subsequently phosphorylate BRCA2 in solution (Figure 2b, lane 1). In contrast, no elution of the kinase was found when we used the mutant GST 18 ± 141 (dD), which is deleted o the cJun homology region (Figure 2b, lane 2). Taken together, our data indicate that, in vitro, BRCA2 is binding to and phosphorylated by a kinase present in Hela nuclear extracts, and that the c-Jun homology region located within exon 3 activation domain constitutes the kinase binding site. Since all JNK family members bind and phosphorylate the d-domain of c-Jun (Gupta et al., 1996), the above-mentioned data still left open the possibility that one member of the JNK family, distinct of JNK1, was the kinase associated with BRCA2 c-Jun homology domain. Activation of JNK protein kinases by exposure to environmental stress, such as UV irradiation, is a characteristic feature of these kinases (Hibi et al., 1993; Gupta et al., 1996). We therefore tested whether the BRCA2-associated kinase is inducible by

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Figure 3 The BRCA2-associated kinase is not induced by UV. U2OS cells were irradiated or not with UV-C (70 J/m2 for 5 s). Unstimulated or UV-stimulated cells were then harvested and lysed in IPH buer (Bannister and Kouzarides, 1996). Whole cell extracts were incubated for 12 h at 48C with equivalent amounts of either GST BRCA2 18 ± 141 (lanes 1 and 2) or, as a positive control, GST c-Jun 30 ± 78 (lanes 3 and 4). Following the solidphase kinase assay, performed as described in Figure 1b, the proteins were separated by SDS ± PAGE Oncogene


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Figure 4 BRCA2 activation domain is phosphorylated in vivo. U2OS cells were transiently transfected with 10 mg of an expression vector containing an Ha-epitope fused to either BRCA2 18 ± 105 (lanes 1 and 2; Ha-BRCA2 18 ± 105) or BRCA2 18 ± 60 (lanes 3 and 4; Ha-BRCA2 18 ± 60). Forty-eight hours after transfection, cells were labelled with 10 mCi of [32P]orthophosphate for 24 h. The cells were then lysed in 500 ml of IPH buer (Bannister and Kouzarides, 1996). Whole cell extracts were precipitated with either anti-Ha antibody (3F10, Boehringer; lanes 2 and 4) or, as a negative control, anti-Flag antibody (M2, Kodak; lanes 1 and 3). Precipitated proteins were separated by SDS ± PAGE and visualized by autoradiography

UV-light. The results shown in Figure 3 indicate that this is not the case. For these experiments, extracts of UV-treated or untreated U2OS cells were incubated with GST fusion proteins, followed by a solid-phase kinase assay. As expected, UV irradiation led to a large increase in the phosphorylation of GST c-Jun (30 ± 78) (Figure 3, compare lanes 3 and 4). In contrast, when we used GST BRCA2 18 ± 141, no signi®cant enhancement of in vitro phosphorylation could be detected from UV-irradiated cells as compared to untreated cells (Figure 3, compare lanes 1 and 2). Thus these results, together with the failure of JNK1 to bind and phosphorylate BRCA2 (see Figure 1b), indicate that BRCA2 is phosphorylated independently of the JNK signalling pathway. The fact that the BRCA2-associated kinase recognizes sequences homologous to the

binding site for JNK does suggest however that this kinase may have structural features analogous to JNK. Having shown in vitro that BRCA2 activation domain is binding to and phosphorylated by a cellular kinase, we next sought to con®rm these results in vivo. To this end, we generated mammalian expression vectors containing either the entire BRCA2 activation domain (residues 18 ± 105) or only residues 18 ± 60 (i.e. lacking its d-homology region), tagged with an Ha epitope. U2OS cells were transfected with these Hatagged BRCA2 constructs and metabolically labelled with 32P-orthophosphate. Following the labelling period, labelled fusion proteins were immunoprecipitated from extracts using anti-Ha antibody. As shown in Figure 4, a 32P-labelled protein of the appropriate molecular-weight was precipitated with anti-Ha antibody in cells transfected with Ha-BRCA2 18 ± 105 (lane 2). No precipitate was detected after substitution of the Ha antibody with the control anti-Flag antibody (lane 1). In contrast to Ha-BRCA2 18 ± 105, no labelled protein was precipitate from cells transfected with HaBRCA2 18 ± 60 which lacks the c-Jun homology region (Figure 4, lanes 3 and 4). Thus, these data indicate that BRCA2 is phosphorylated in vivo and, consistent with the in vitro results presented in Figure 2, the exon 3 activation domain is required for BRCA2 phosphorylation. In summary, we show that the BRCA2 activation domain associates with a cellular kinase which can phosphorylate the BRCA2 activation domain. The binding site for the BRCA2-associated kinase corresponds to residues which have homology to the JNK binding site in c-Jun. While the nature of the BRCA2associated kinase is currently unknown, the present data provide insights into the regulation of the BRCA2 protein and suggest that phosphorylation may be one mechanism by which BRCA2's transcriptional function is regulated.

Acknowledgments J Milner was supported by a grant from the Cancer Research Campaign; F Fuks was supported by fellowships from the European Community Training and Mobility of Researchers Fellowship and the Wiener-Anspach Fundation; L Hughes-Davies was funded by a CRC Clinical Fellowship.

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