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The BRCT Domain Is a Phospho-Protein Binding Domain

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Science  24 Oct 2003:
Vol. 302, Issue 5645, pp. 639-642
DOI: 10.1126/science.1088753

Abstract

The carboxyl-terminal domain (BRCT) of the Breast Cancer Gene 1 (BRCA1) protein is an evolutionarily conserved module that exists in a large number of proteins from prokaryotes to eukaryotes. Although most BRCT domain–containing proteins participate in DNA-damage checkpoint or DNA-repair pathways, or both, the function of the BRCT domain is not fully understood. We show that the BRCA1 BRCT domain directly interacts with phosphorylated BRCA1-Associated Carboxyl-terminal Helicase (BACH1). This specific interaction between BRCA1 and phosphorylated BACH1 is cell cycle regulated and is required for DNA damage–induced checkpoint control during the transition from G2 to M phase of the cell cycle. Further, we show that two other BRCT domains interact with their respective physiological partners in a phosphorylation-dependent manner. Thirteen additional BRCT domains also preferentially bind phospho-peptides rather than nonphosphorylated control peptides. These data imply that the BRCT domain is a phospho-protein binding domain involved in cell cycle control.

The BRCT domain was first identified as ∼100 –amino acid tandem repeats at the C-terminus of the tumor-suppressor gene product BRCA1, in which the germline mutations lead to ∼50% familial breast cancer. A large number of proteins also contain single or multiple BRCT motifs, and many of them appear to function in DNA-damage checkpoint control and DNA repair (1, 2).

The BRCT domains are important for the tumor-suppressor function of BRCA1. Most BRCA1 mutations cause truncated BRCA1 gene products that lack one or both C-terminal BRCT domains, and clinically relevant missense mutations identified at the C-terminus of BRCA1 abolish the BRCT structure (3). Loss of the BRCA1 BRCT domains leads to tumor formation in mice (4). Studies on other BRCT-containing proteins also suggest that the BRCT domain may directly participate in DNA-damage responses, cell cycle checkpoints, or DNA repair (58).

Thus, the BRCT domain is an important motif involved in cellular responses to DNA damage. We reasoned that the BRCT domain might be directly involved in signal transduction. To explore this possibility, we examined the interaction between BRCA1 and BACH1, which is a putative DEAH helicase interacting with the BRCA1 BRCT domains (9). We first examined whether the interaction between the BRCA1 BRCT domain and BACH1 depends on the phosphorylation status of BACH1.

We generated recombinant BRCA1-BRCT domains (residues 1599 to 1863) fused to glutathione S-transferase (GST). The BRCA1 BRCT domain bound endogenous BACH1 from a 293T human kidney cell lysate (Fig. 1A) (9). Inclusion of phosphatase inhibitors (10 mM NaF and 50 mM β-glycerophosphate) in the lysis buffer increased the interaction between BRCA1 BRCT and BACH1, and lysates that had first been treated with λ protein phosphatase (PPase) abolished this interaction (Fig. 1A). Because λ PPase treatment had no effect on the amount of BACH1, these results suggest that the BRCA1-BACH1 interaction depends on the phosphorylation of BACH1.

Fig. 1.

Dependence of the BRCA1-BACH1 interaction on the phospho-Ser990 of BACH1. (A) Whole-cell lysates prepared from 293T cells were treated with or without phosphatase inhibitor or λ phosphatase (PPase), and then incubated with GST-BRCA1-BRCT protein immobilized on Sepharose beads. Proteins bound to beads were eluted and separated by SDS–polyacrylamide gel electrophoresis. Western blots were done with antibody to BACH1 (upper panel). Western blots of whole-cell lysates demonstrated equal amounts of BACH1 in these samples (middle panel). The equal loading of GST proteins was confirmed with monoclonal antibody to GST (lower panel). Myc epitope–tagged BACH1 deletion mutants (B) or single–amino acid substitution mutants (C) were transiently expressed in 293T cells. Cell lysates were incubated with beads containing GST-BRCA1-BRCT proteins. Associated BACH1 proteins were detected with anti-myc (upper panels). Western blots with anti-myc on the whole-cell lysates ensured similar expression levels of various BACH1 mutants in these samples (middle panels).

A region between residues 888 and 1063 of BACH1 is responsible for the interaction between BACH1 and BRCA1 (9), suggesting that this region may contain the potential BACH1 phosphorylation sites required for its interaction with the BRCA1 BRCT domain. Deletion of residues 980 to 1018 of BACH1 prevented the interaction of BACH1 with the BRCA1 BRCT domain (Fig. 1B). In this region of BACH1, there are 12 serine, threonine, and tyrosine sites that could be the potential phosphorylation sites. We mutated each of these residues to alanine. Pull-down experiments demonstrated that only Ser990 of BACH1 was essential for the BACH1-BRCA1 interaction (Fig. 1C). To confirm this observation in the cell, wild-type BACH1 or a Ser990-to-alanine (S990A) mutant of BACH1 was expressed in 293T cells. Only wild-type BACH1, but not the S990A mutant, coimmunoprecipitated with endogenous BRCA1, confirming that the BRCA1-BACH1 interaction requires Ser990 of BACH1 (Fig. 2A).

Fig. 2.

In vivo recognition of phosphorylated Ser990 of BACH1 by BRCA1. (A and B) Myc epitope–tagged full-length BACH1 or S990A mutant was transiently expressed in 293T cells. Immunoprecipitation and immunoblotting experiments were done with anti-myc or anti-BRCA1 as indicated (A). (B) Proteins from cell lysates were immunoprecipitated with anti-myc and immunoblotted with anti-(p)S990 (upper panel) and anti-BACH1 (lower panel). (C) 293T cell lysates were treated with or without phosphatase inhibitor, in the absence or presence of λ phosphatase, and then immunoprecipitated with anti-BACH1 and blotted with anti-(p)S990 (upper panel). Whole-cell lysates were blotted with anti-BACH1 (lower panel). (D) BACH1 phosphorylation and the BRCA1-BACH1 interaction are cell cycle regulated. HeLa cells were arrested in M phase by treatment with nocodazole (1 μg/ml) for 24 hours, and then incubated in fresh medium for the indicated time. Cell lysates were analyzed by immunoprecipitation and immunoblotting as indicated. Western blot with Chk2 was included as a protein loading control because Chk2 expression is not cell cycle regulated. Results of a cell cycle profile examined by flow cytometric analysis were also included.

To test whether Ser990 of BACH1 is phosphorylated in vivo, we generated a phospho-specific antibody to a peptide containing phospho-Ser990 of BACH1. This antibody specifically recognized wild-type BACH1 in cell lysates, but not the S990A mutant (Fig. 2B). Although this antibody recognized endogenous BACH1, it failed to recognize BACH1 that had first been treated with λ PPase (Fig. 2C). A phosphatase inhibitor reversed the effect of λ PPase. Taken together, these results suggest that Ser990 of BACH1 is phosphorylated in vivo.

The residue following Ser990 of BACH1 is a proline. Phosphorylation of sites containing serine or threonine followed by proline is usually regulated by cyclin-dependent kinases. In addition, the expression and phosphorylation of BRCA1 are regulated when G0 cells enter the cell cycle (10). These observations raised the possibility that the phosphorylation-dependent interaction between BRCA1 and BACH1 might be regulated in a cell cycle–dependent manner. To test this possibility, we arrested HeLa cells in mitosis and released them into the cell cycle. Amounts of BRCA1 decreased slightly during G1 phase, whereas expression of BACH1 was stable throughout the cell cycle (Fig. 2D). However, Ser990 of BACH1 was phosphorylated from S to G2-M phase, but not in G1 cells (Fig. 2D). We also observed an increased interaction between BRCA1 and BACH1 as cells progressed from S to G2-M phase (Fig. 2D). In contrast, the BRCA1-BACH1 complex was not detected in G1 phase, although both BRCA1 and BACH1 were expressed in G1 cells. Thus, the phosphorylation of BACH1 and its interaction with the BRCA1 BRCT domain are cell cycle regulated.

Normal cells treated with ionizing radiation arrest in the G2 phase of the cell cycle and do not enter mitosis until DNA repair is completed. This G2-M checkpoint control is absent in BRCA1-deficient cells (1113). To examine whether the interaction between BRCA1 and BACH1 is required for G2-M checkpoint control, we used HCC1937 human breast carcinoma cells, which synthesize a truncated form of BRCA1 that lacks one of the BRCT domains. As expected, the truncated mutant BRCA1 failed to associate with BACH1, whereas wild-type BRCA1 did interact with BACH1 (fig. S1) (9). We also observed a G2-M checkpoint defect in HCC1937 cells, which was restored upon introduction of wild-type BRCA1 (Fig. 3A). This suggests a critical role for the C-terminal BRCT domains in the maintenance of the G2-M checkpoint.

Fig. 3.

Requirement of BRCA1-BACH1 interaction for G2-M checkpoint control. (A) Defective G2-M checkpoint control in HCC1937 cells. G2-M checkpoint assays were done as described (16). The fraction of cells in M phase is expressed as a percentage of that measured in un-irradiated control cells. (B) A549 cells were treated with SM or SPM lentivirus, followed by BACH1 siRNA transfection. G2-M checkpoint assays were performed 48 hours later.

To study specifically the BRCA1-BACH1 interaction in vivo, we generated BACH1 small interfering RNA (siRNA) that decreases BACH1 expression when transfected into A549 cells (fig. S2). γ-Ray–irradiated A549 cells transfected with BACH1 siRNA failed to arrest in G2 and accumulated in M phase (Fig. 3B), suggesting that, like BRCA1, BACH1 is required for damage-induced G2-M checkpoint control. The expression of an siRNA-resistant BACH1 (SM) fully restored G2-M checkpoint control, but a SM plus S990A BACH1 mutant (SPM) that does not interact with BRCA1 failed to rescue the G2-M checkpoint defect (Fig. 3B). These data indicate that the phosphorylation-dependent interaction between BACH1 and the BRCA1 BRCT domains is important for the activation of G2/M checkpoint.

We synthesized peptides corresponding to residues 985 to 998 of BACH1, which contained either phosphorylated or unphosphorylated (control) Ser990. Only the phospho-peptide competed with endogenous BACH1 for binding to the BRCA1 BRCT domain (Fig. 4A), further suggesting that BRCA1 binds to the phosphorylated Ser990 site of BACH1. We also examined the direct interaction between the BACH1 peptides and the BRCA1 BRCT domain using surface plasmon resonance. The BRCA1-BRCT domain did not bind to unphosphorylated peptide, but it bound tightly to the phosphorylated peptide (Fig. 4B). The dissociation constant (KD) was 113 nM. The KD for phospho-BACH1 and recombinant BRCA1 BRCT domains lacking a GST tag was similar (68 nM). Using isothermal titration calorimetry, we also determined the affinity between the BRCA1 BRCT domain and BACH1 phosphopeptide to be 2.4 μM (fig. S3). Such a discrepancy between these measurements has been reported previously (14, 15). Two BRCA1 BRCT domain missense mutants, P1749R and M1775R, also failed to interact with the BACH1 phospho-peptide (Fig. 4B), suggesting that the intact structure of the tandem BRCA1 BRCT domains is essential for its interaction with the phosphorylated substrate.

Fig. 4.

High-affinity binding of the BRCA1 BRCT domain to phosphorylated BACH1 peptides. (A) Peptide competition assay. 293T cell lysates were incubated with beads containing GST-BRCA1-BRCT proteins in the presence of5 μM phosphorylated peptide [(p)S: VISRST(p)SPTFNKQTC] or unphosphorylated control peptide (S: VISRSTSPTFNKQTC). Associated BACH1 was detected with antibodies to BACH1. (B) GST BRCA1-BRCT fusion protein (2 nM to 2 μM), mutant BRCA1 BRCT domains (2 nM to 2 μM), or BRCA1-BRCT protein (1 nM to 3 μM) were passed over BIAcore chip surfaces conjugated with control peptide (S990: ISRSTSPTFNKQTK-biotin) or phosphorylated peptide [(p)S 990 peptide: ISRST(p)SPTFNKQTK-biotin]. Resonance units were measured by BIAcore 3000. (C) Single–amino acid substitution mutants were transiently expressed in 293T cells. Cell lysates were incubated with beads containing GST-BRCA1-BRCT protein. Associated BACH1 proteins were detected with anti-myc (upper panel) or anti-(p)S990 BACH1 (middle panel). (D) Relative binding of BRCA1 BRCT domain with indicated phospho-peptides containing single–amino acid substitutions conjugated to the surface of Biacore chips.

In addition to the phospho-Ser990 in BACH1, other surrounding residues may also contribute to the BRCA1-BACH1 interaction. Serines at the –4 and –2 positions and threonines at the –1, +2, and +7 positions are not important for the BRCA1-BACH1 interaction (Fig. 1C). We sequentially mutated all the remaining residues between the –6 and +6 positions to alanine. Only Pro991-to-Ala (P991A) and Phe993-to-Ala (F993A) disrupted the BRCA1 BRCT domain–BACH1 interaction. However, both mutants also abolished the phosphorylation of Ser990 in vivo (Fig. 4C). Whereas phospho-peptides containing P991A or T992A mutations had slightly reduced affinity for the BRCA1 BRCT domain, the F993A phospho-peptide did not bind the BRCA1 BRCT domain in vitro (Fig. 4D), indicating that the +3 position is the major determinant of the binding specificity for BRCA1 BRCT domains.

To further demonstrate that the binding of phospho-peptides is a general characteristic of BRCT domains, we examined the BRCT domain of Fcp1 and one of the BRCT domains of Top BP1 (BRCT6). Fcp1 BRCT domain and TopBP1 BRCT6 bound to phosphorylated RNA polymerase II and E2F1, respectively (fig. S5). We also synthesized a degenerate phospho-peptide library based on the sequence of BACH1. Ten additional tandem BRCT domains and three single BRCT domains preferentially bound phospho-peptides (fig. S6). Again, the integrity of BRCT domains is critical for these phospho-specific interactions, because mutations of the highly conserved Trp in these BRCT domains abolished their abilities to interact with phospho-targets (figs. S4 and S5).

In this study, we have provided several lines of evidence suggesting that the BRCT domain is a phospho-protein recognition motif. First, we have shown that the BRCA1 BRCT domain directly recognizes the phosphorylated Ser990 site of BACH1. In addition, other BRCT domain–containing proteins such as TopBP1 and Fcp1 bind to their physiological partners in a phospho-dependent manner. Ten additional tandem BRCT domains and three single BRCT domains all preferentially bind phosphopeptides. Thus, our results unveil an important aspect of these BRCT domains: The BRCT domain is a phospho-protein binding motif. Given that BRCT domain–containing proteins are involved in multiple cellular responses, including cell cycle checkpoint control, DNA repair, and transcription regulation, understanding the interaction between BRCT domains and their physiological targets will help us appreciate the complex regulation of these cellular responses.

Supporting Online Material

www.sciencemag.org/cgi/content/full/302/5645/639/DC1

Materials and Methods

Figs. S1 to S6

References and Notes

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