BRCA1 tumor suppression depends on BRCT phosphoprotein binding, but not its E3 ligase activity

Abstract

Germline mutations of the breast cancer 1 (BRCA1) gene are a major cause of familial breast and ovarian cancer. The BRCA1 protein displays E3 ubiquitin ligase activity, and this enzymatic function is thought to be required for tumor suppression. To test this hypothesis, we generated mice that express an enzymatically defective Brca1. We found that this mutant Brca1 prevents tumor formation to the same degree as does wild-type Brca1 in three different genetically engineered mouse (GEM) models of cancer. In contrast, a mutation that ablates phosphoprotein recognition by the BRCA C terminus (BRCT) domains of BRCA1 elicits tumors in each of the three GEM models. Thus, BRCT phosphoprotein recognition, but not the E3 ligase activity, is required for BRCA1 tumor suppression.

Fig. 1

The enzymatic activity of Brca1 is dispensable for tumor suppression. (A) Kaplan-Meier tumor-free survival curves of Kras^LSL-G12D/p53^flex7/flex7/Pdx1-cre (KPC-Brca1^+/+; black curve; T₅₀ = 68 days) mice compared with Kras^LSL-G12D/p53^flex7/flex7/Pdx1-cre/Brca1^flex2/flex2 (KPC-Brca1^flex2/flex2; blue curve; T₅₀ = 40 days; P < 0.0001) and Kras^LSL-G12D/p53^flex7/flex7/Pdx1-cre/Brca1^{flex2/FH-I26A} (KPC-Brca1^{flex2/FH-I26A}; red curve; T₅₀ = 65 days; P = 0.2595) mice. (B) Kaplan-Meier survival curves of Brca1^flex2/flex2/Wap^cre/+ (C-Brca1^flex2/flex2; green curve; T₅₀ = 512 days) females compared with Brca1^{flex2/FH-I26A}/Wap^cre/+ (C-Brca1^{flex2/FH-I26A}; red curve; P < 0.0001) females. (C) Kaplan-Meier survival curves of p53^LSL-R270H/+/Wap^cre/+ (PC-Brca1^+/+; black curve; T₅₀ = 380 days) females compared with Brca1^flex2/flex2/p53^LSL-R270H/+/Wap^cre/+ (PC-Brca1^flex2/flex2; blue curve; T₅₀ = 309 days; P < 0.0001) and Brca1^{flex2/FH-I26A}/p53^LSL-R270H/+/Wap^cre/+ (PC-Brca1^{flex2/FH-I26A}; red curve; T₅₀ = 382 days; P = 0.5354) females. (D) Kaplan-Meier survival curves of control Brca1^FH-WT/FH-WT (black curve) mice compared with Brca1^tr/tr (blue curve; T₅₀ = 529 days; P < 0.0001) and Brca1^{FH-I26A/FH-I26A} and Brca1^FH-I26A/– (red curve; P = 0.5197) mice.

Fig. 2

The BRCT phospho-recognition property of Brca1 is critical for the DNA damage response. (A) S1598F ablates the interaction between Bach1/FancJ and endogenous Brca1. MEFs were prepared from mice with a knock-in allele (Bach1^WT-FH) encoding wild-type Bach1 with C-terminal Flag-hemagglutinin (HA) epitopes. Bach1^WT-FH and Brca1 levels were examined by immunoblotting lysates of Brca1^+/+/Bach1^WT-FH/+ and Brca1^{S1598F/S1598F}/Bach1^WT-FH/+ MEFs (left). To evaluate the Brca1/Bach1^WT-FH interaction, lysates were immunoprecipitated with a Brca1-specific antiserum or corresponding preimmune serum (pre) and immunoblotted with HA-specific antibodies (right). The input (left) represents 2.5% of the protein amount used for immunoprecipitation (right). (B) Brca1^{S1598F/S1598F} cells are sensitive to genotoxic stress. ES cells proficient (Brca1^+/− and Brca1^S1598F/+) or deficient (Brca1^{S1598F/S1598F}) for BRCT phospho-recognition were examined for mitomycin C (MMC) sensitivity in clonogenic survival assays, together with ES cells homozygous for the hypomorphic Brca1^Δ223-763 mutation. (C) BRCT phospho-recognition by Brca1 is essential for homology-directed DNA repair. Brca1^+/−, Brca1^{S1598F/S1598F}, and Brca1^{Δ223-763/Δ223-763} ES subclones containing the direct repeat–green fluorescent protein (DR-GFP) substrate integrated into the Pim1 locus were transfected with either an I-SceI expression vector or empty vector. I-SceI strongly induced the number of GFP-positive cells in Brca1^+/− cells (clone 4), indicating efficient homology-directed repair, but not in Brca1^{S1598F/S1598F} cells (clones 1 to 3). The reduction in GFP-positive cells in Brca1^{S1598F/S1598F} cells was similar to that of Brca1^{Δ223-763/Δ223-763} ES cells (clone 5), which are known to be deficient in homology-directed repair (28).

Fig. 3

BRCT phospho-recognition is essential for Brca1 tumor suppression. (A) Kaplan-Meier tumor-free survival curves of p53^LSL-R270H/+/Wap^cre/+ (PC-Brca1^+/+; black curve; T₅₀ = 380 days) females compared with Brca1^flex2/flex2/p53^LSL-R270H/+/Wap^cre/+ (PC-Brca1^flex2/flex2; blue curve; T₅₀ = 309 days; P < 0.0001) and Brca1^flex2/S1598F/p53^LSL-R270H/+/Wap^cre/+ (PC-Brca1^flex2/S1598F; green curve; T₅₀ = 308 days; P < 0.0001) females. (B) Kaplan-Meier survival curves of Kras^LSL-G12D/p53^flex7/flex7/Pdx1-cre (KPC-Brca1^+/+; black curve; T₅₀ = 68 days) mice compared with Kras^LSL-G12D/p53^flex7/flex7/Pdx1-cre/Brca1^flex2/flex2 (KPC-Brca1^flex2/flex2; blue curve; T₅₀ = 40 days; P < 0.0001) and Kras^LSL-G12D/p53^flex7/flex7/Pdx1-cre/Brca1^flex2/S1598F (KPC-Brca1^flex2/S1598F; green curve; T₅₀ = 45 days; P < 0.0001) mice. (C) Kaplan-Meier survival curves of Brca1^FH-WT/FH-WT (black curve) mice compared with Brca1^tr/tr (blue curve; P < 0.0001) and Brca1^{S1598F/S1598F} (green curve; P < 0.0001) mice.