RESUMEN
BARD1 is a breast cancer tumor suppressor with multiple domains and functions. BARD1 comprises a tandem BRCT domain at the C-terminus, and this sequence has been reported to target BARD1 to distinct subcellular locations such as nuclear DNA breakage sites and the centrosome through binding to regulatory proteins such as HP1 and OLA1, respectively. We now identify the BRCT domain as a binding site for p53. We first confirmed previous reports that endogenous BARD1 binds to p53 by immunoprecipitation assay, and further show that BARD1/p53 complexes locate at mitochondria suggesting a cellular location for p53 regulation of BARD1 apoptotic activity. We used a proximity ligation assay to map three distinct p53 binding sequences in human BARD1, ranging from weak (425-525) and modest (525-567) to strong (551-777 comprising BRCT domains). Deletion of the BRCT sequence caused major defects in the ability of BARD1 to (1) bind p53, (2) localize to the cytoplasm and mitochondria, and (3) induce Bax oligomerization and apoptosis. Our data suggest that BARD1 can move to mitochondria independent of p53, but subsequently associates with p53 to induce Bax clustering in part by decreasing mitochondrial Bcl-2 levels. We therefore identify a role for the BRCT domain in stimulating BARD1 nuclear export and mitochondrial localization, and in assembling mitochondrial BARD1/p53 complexes to regulate specific activities such as apoptotic function.
Asunto(s)
Apoptosis , Neoplasias de la Mama/metabolismo , Citoplasma/metabolismo , Mitocondrias/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Secuencia de Aminoácidos , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Citoplasma/genética , Roturas del ADN , Femenino , Humanos , Células MCF-7 , Mitocondrias/genética , Mitocondrias/patología , Estructura Terciaria de Proteína , Transporte de Proteínas/genética , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Eliminación de Secuencia , Proteína p53 Supresora de Tumor/genética , Proteínas Supresoras de Tumor/genética , Ubiquitina-Proteína Ligasas/genética , Proteína X Asociada a bcl-2/genética , Proteína X Asociada a bcl-2/metabolismoRESUMEN
The inositol 1,4,5-trisphosphate receptor (IP3R) is a ubiquitously expressed endoplasmic reticulum (ER)-resident calcium channel. Calcium release mediated by IP3Rs influences many signaling pathways, including those regulating apoptosis. IP3R activity is regulated by protein-protein interactions, including binding to proto-oncogenes and tumor suppressors to regulate cell death. Here we show that the IP3R binds to the tumor suppressor BRCA1. BRCA1 binding directly sensitizes the IP3R to its ligand, IP3. BRCA1 is recruited to the ER during apoptosis in an IP3R-dependent manner, and, in addition, a pool of BRCA1 protein is constitutively associated with the ER under non-apoptotic conditions. This is likely mediated by a novel lipid binding activity of the first BRCA1 C terminus domain of BRCA1. These findings provide a mechanistic explanation by which BRCA1 can act as a proapoptotic protein.
Asunto(s)
Apoptosis , Proteína BRCA1/metabolismo , Calcio/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Señalización del Calcio , Línea Celular Tumoral , Retículo Endoplásmico/metabolismo , Humanos , Modelos Moleculares , Neoplasias/metabolismoRESUMEN
BRCA1 is a DNA damage response protein and functions in the nucleus to stimulate DNA repair and at the centrosome to inhibit centrosome overduplication in response to DNA damage. The loss or mutation of BRCA1 causes centrosome amplification and abnormal mitotic spindle assembly in breast cancer cells. The BRCA1-BARD1 heterodimer binds and ubiquitinates γ-tubulin to inhibit centrosome amplification and promote microtubule nucleation; however regulation of BRCA1 targeting and function at the centrosome is poorly understood. Here we show that both N and C termini of BRCA1 are required for its centrosomal localization and that BRCA1 moves to the centrosome independently of BARD1 and γ-tubulin. Mutations in the C-terminal phosphoprotein-binding BRCT domain of BRCA1 prevented localization to centrosomes. Photobleaching experiments identified dynamic (60%) and immobilized (40%) pools of ectopic BRCA1 at the centrosome, and these are regulated by the nuclear export receptor CRM1 (chromosome region maintenance 1) and BARD1. CRM1 mediates nuclear export of BRCA1, and mutation of the export sequence blocked BRCA1 regulation of centrosome amplification in irradiated cells. CRM1 binds to undimerized BRCA1 and is displaced by BARD1. Photobleaching assays implicate CRM1 in driving undimerized BRCA1 to the centrosome and revealed that when BRCA1 subsequently binds to BARD1, it is less well retained at centrosomes, suggesting a mechanism to accelerate BRCA1 release after formation of the active heterodimer. Moreover, Aurora A binding and phosphorylation of BRCA1 enhanced its centrosomal retention and regulation of centrosome amplification. Thus, CRM1, BARD1 and Aurora A promote the targeting and function of BRCA1 at centrosomes.
Asunto(s)
Proteína BRCA1/metabolismo , Neoplasias de la Mama/metabolismo , Núcleo Celular/metabolismo , Centrosoma/metabolismo , Carioferinas/metabolismo , Señales de Exportación Nuclear , Proteínas Serina-Treonina Quinasas/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Transporte Activo de Núcleo Celular , Aurora Quinasas , Proteína BRCA1/genética , Neoplasias de la Mama/genética , Línea Celular Tumoral , Núcleo Celular/genética , Daño del ADN/genética , Femenino , Humanos , Carioferinas/genética , Mutación , Fosforilación , Unión Proteica , Proteínas Serina-Treonina Quinasas/genética , Estructura Terciaria de Proteína , Receptores Citoplasmáticos y Nucleares/genética , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Proteína Exportina 1RESUMEN
BARD1 heterodimerizes with BRCA1, forming an E3 ubiquitin ligase that functions at nuclear foci to repair DNA damage and the centrosome to regulate mitosis. We compared BARD1 recruitment at these structures using fluorescence recovery after photobleaching assays to measure YFP-BARD1 dynamics in live cells. In nuclei at ionizing radiation-induced foci, 20% of the BARD1 pool was immobile and 80% of slow mobility exhibiting a recovery time >500 s. In contrast, at centrosomes 83% of BARD1 was rapidly mobile with extremely fast turnover (recovery time ~20s). The ~25-fold faster exchange of BARD1 at centrosomes correlated with BRCA1-independent recruitment. We mapped key targeting sequences to a combination of the N and C-termini, and showed that mutation of the nuclear export signal reduced centrosome localization by 50%, revealing a role for CRM1. Deletion of the sequence 128-550 increased BARD1 turnover at the centrosome, consistent with a role in transient associations. Conversely, the cancer mutation Q564H reduced turnover by 25%. BARD1 is one of the most highly mobile proteins yet detected at the centrosome, and in contrast to its localization at DNA repair foci, which requires dimerization with BRCA1, targeting of BARD1 to the centrosome occurs prior to heterodimerization and its rapid turnover may provide a mechanism to regulate dimer formation.
Asunto(s)
Proteína BRCA1/metabolismo , Neoplasias de la Mama/metabolismo , Neoplasias del Colon/metabolismo , Carioferinas/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Transducción de Señal , Proteínas Supresoras de Tumor/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Transporte Activo de Núcleo Celular/genética , Transporte Activo de Núcleo Celular/efectos de la radiación , Proteína BRCA1/genética , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Línea Celular Tumoral , Núcleo Celular/genética , Núcleo Celular/metabolismo , Centrosoma/metabolismo , Centrosoma/efectos de la radiación , Neoplasias del Colon/genética , Neoplasias del Colon/patología , Daño del ADN/efectos de la radiación , Reparación del ADN/efectos de la radiación , Dimerización , Fluorescencia , Recuperación de Fluorescencia tras Fotoblanqueo , Rayos gamma , Humanos , Carioferinas/genética , Mitosis/efectos de la radiación , Mutación , Plásmidos , Receptores Citoplasmáticos y Nucleares/genética , Transducción de Señal/efectos de la radiación , Transfección , Proteínas Supresoras de Tumor/química , Proteínas Supresoras de Tumor/genética , Ubiquitina-Proteína Ligasas/química , Ubiquitina-Proteína Ligasas/genética , Proteína Exportina 1RESUMEN
The BRCA1/BARD1 heterodimer regulates genomic maintenance and contributes to the DNA damage checkpoint response. We previously reported that BRCA1 and BARD1 can shuttle between nucleus and cytoplasm. In this study, we evaluated the localisation patterns of BRCA1 and BARD1 in response to different types of DNA damaging agents and chemotherapeutic drugs. In MCF-7 cells, endogenous BRCA1 increased transiently in the nucleus at 2h after ionising radiation (IR), whereas BARD1 was unaffected. IR treatment did not induce nuclear export of either protein, in contrast to previous reports. DNA damage by UV radiation, etoposide or camptothecin caused a preferential down-regulation of nuclear BARD1 at 6h post-treatment. The UV-dependent loss of nuclear BARD1 was blocked by the proteasome inhibitor MG132, but not by leptomycin B, indicating a change in BARD1 nuclear degradation rather than nuclear export. MG132 also blocked the dispersal of BARD1/BRCA1 nuclear foci at 6h after UV, implicating the proteasome in repair foci disassembly. In the cytoplasm, BRCA1 and BARD1 were detected at centrosomes but their distribution was not altered by DNA damage. BARD1 displayed a stronger mitochondria accumulation than BRCA1, and became phosphorylated at mitochondria in response to DNA damage. The mitotic spindle poisons vincristine and paclitaxel had no effect on BRCA1 or BARD1 subcellular distribution. We conclude that BARD1 phosphorylation, expression and localisation patterns are regulated in the nucleus and at mitochondria in response to different forms of DNA damage, contributing to the role of BRCA1/BARD1 in DNA repair and apoptotic responses.