Characterization of BRCA1 Protein Targeting, Dynamics, and Function at the Centrosome

• Published in: The Journal of biological chemistry 2012-03, Vol.287 (10), p.7701-7716
• Main Authors: Brodie, Kirsty M., Henderson, Beric R.
• Format: Article
• Language: English
• Subjects: Aurora A Kinase; BARD1; BRCA1; Cell Biology; Centrosome; Centrosome Amplification; Confocal Microscopy; CRM1; FRAP Assay; Protein Targeting
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M111.327296

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. BRCA1 inhibits centrosome duplication as part of the DNA damage checkpoint. Binding partners CRM1, BARD1, and Aurora A have distinct roles in targeting BRCA1 to the centrosome and regulating its activity. BRCA1 nuclear export stimulates its regulation of centrosome duplication. CRM1 mediates shuttling of BRCA1 between nucleus and centrosome and may coordinate its DNA damage response.