title: Characterization of BRCA1 protein targeting, dynamics, and function at the centrosome: a role for the nuclear export signal, CRM1, and Aurora A kinase.
abstract: 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.
authors.affiliation: Westmead Institute for Cancer Research, University of Sydney, Darcy Road (P.O. Box 412), Westmead, New South Wales 2145, Australia.
authors.forename: Kirsty M
authors.initials: KM
authors.lastname: Brodie
authors.forename: Beric R
authors.initials: BR
authors.lastname: Henderson
mesh_terms: D021581:Active Transport, Cell Nucleus; D064127:Aurora Kinases; D019313:BRCA1 Protein / Q000235:genetics / Q000378:metabolism*; D001943:Breast Neoplasms / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000235:genetics / Q000378:metabolism*; D018385:Centrosome / Q000378:metabolism*; D004249:DNA Damage / Q000235:genetics; D005260:Female; D006801:Humans; D028884:Karyopherins / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D049790:Nuclear Export Signals; D010766:Phosphorylation; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics / Q000378:metabolism*; D014404:Tubulin / Q000235:genetics / Q000378:metabolism; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism; D000097606:Exportin 1 Protein
chemical_list: D064127:Aurora Kinases; D019313:BRCA1 Protein; D028884:Karyopherins; D049790:Nuclear Export Signals; D017346:Protein Serine-Threonine Kinases; D018160:Receptors, Cytoplasmic and Nuclear; D014404:Tubulin; D025521:Tumor Suppressor Proteins; D044767:Ubiquitin-Protein Ligases; D000097606:Exportin 1 Protein; C492913:BRCA1 protein, human; C102449:BARD1 protein, human