In vitro phosphorylation of BRCA2 by the checkpoint kinase CHEK2

Abstract

Germline mutations in both BRCA2 and CHEK2 are associated with an increased risk for male breast cancer. To search for potential interactions between the products of these breast cancer susceptibility genes, we undertook systematic mapping of BRCA2 for potential phosphorylation sites by CHEK2. In vitro kinase assays and mass spectrometric analysis identified a 50 amino-acid fragment within the N-terminus of BRCA2 potentially targeted by CHEK2, containing two major phosphopeptides. Inducible overexpression of this peptide, but not a derivative with mutated phosphorylation sites, leads to increased chromosome fragmentation and suppression of cellular proliferation. These results suggest a link between CHEK2 and BRCA2 pathways, which may contribute to the spectrum of cancers associated with germline CHEK2 mutations.

Figure 1

Phosphorylation of the N-terminal fragment of BRCA2 (B2P) by CHEK2 in vitro. Schematic representation of BRCA2 in seven overlapping GST-fusion proteins, designated 1–7, and used for in vitro kinase assays. Fragment 1 was further subdivided into the three overlapping GST-fusion proteins, 1.1, 1.2, and 1.3, and the fragment 1.3 region was divided again into fragments 1.3.1, 1.3.2, 1.3.3, and 1.3.4. The final peptide containing specific CHEK2 phosphorylation sites was designated B2P: it has 50 amino acids with four putative substrate sites (shown in red), identified by mass-spectrometric analysis. The mass-spectrometric analysis was performed by using fragments of BRCA2 expressed from bacteria as GST-fusion proteins with either bacterially expressed GST-CHEK2 or immunoprecipitated Flag-CHEK2 from IR-treated tissue culture cells. The right panel shows the in vitro CHEK2 phosphorylation assay for each step of analysis with BRCA2 fragments. GST-BRCA1 fragment containing S988 and GST alone were used as positive and negative controls.

Figure 2

BRCA2 residues targeted by CHEK2 phosphorylation. (A) The four residues containing putative phosphorylation sites in the BRCA2 fragment B2P were mutagenised individually or in combination as shown. (B) Phosphorylation of wild type (WT) or mutant forms of B2P fragments by CHEK2. GST-fusion proteins were incubated with bacterially expressed GST-CHEK2 proteins in the presence of [γ-³²P]ATP. This in vitro kinase assay was repeated three times with similar results. (C) Phosphorylation of B2P by immunoprecipitated CHEK2, following in vivo activation by ionising radiation. Flag-tagged CHEK2 protein was induced for 24 h in U2OS cells with tetracycline-regulated expression, and lysates were collected 1 h following treatment with 10 Gy and immunoprecipitated using either anti-Flag or nonspecific (control) antibodies. Wild-type (WT) or mutant (M4, M9) GST-fusion B2P fragments were incubated with the immunoprecipitates in the presence of ³²P. Phosphorylation of wild-type, but not mutant, GST-BRCA2 fragments is evident in CHEK2 immunoprecipitates. Flag-CHEK2 itself shows autophosphorylation as well. Total lysates (10 μg) were immunoblotted to show comparable expression of Flag-CHEK2 (western blot control).

Figure 3

Suppression of cellular proliferation and induction of chromosome fragmentation by overexpression of wild-type B2P. (A) Inducible expression of B2P. Immunoblotting (α-Flag) analysis of cells with inducible expression of the wild-type Flag-B2P-NLS (B2P-WT), mutant form (B2P-M9), or the known BRCA2 inhibitory peptide BRC4 (actin loading control). (B) Inhibition of cell proliferation by expression of B2P. Cells with inducible expression of B2P, mutant (B2P-M9), BRC4 or vector were grown in the presence (−Ind) or absence (+Ind) of tetracycline. Viable cells were counted at the indicated times. Experiments were performed in triplicate with standard deviation shown. (C) Increased chromosome fragmentation following induction of B2P. The mean number of chromosome fragments per metaphase spread (N=50) is shown for cells with inducible expression of B2P-WT, mutant (B2P-M9), BRC4 or vector alone. The mean fragment/metaphase levels between B2P-M9 and BRC4 were significantly different (P<0.0001). (D) Representative metaphase spread from cells expressing either B2P-WT, B2P-M9, or BRC4. Arrowheads indicate fragmented chromosomes.