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.
Highlights
breast and ovarian cancer susceptibility protein 1 (BRCA1) inhibits centrosome duplication as part of the DNA damage checkpoint
Cancer Mutations in BRCT Domains Abolish Centrosomal Localization of BRCA1—Because centrosomal localization is integral to BRCA1-dependent regulation of centrosome amplification and tumor suppressor function, we compared the effect of known cancer-associated mutations on the localization of BRCA1 to the centrosome
This could be due to the fact that such point mutations disrupt folding of the BRCT domain region [45] and thereby prevent binding of specific proteins required for recruitment of BRCA1 to the centrosome
Summary
BRCA1 inhibits centrosome duplication as part of the DNA damage checkpoint. Results: Binding partners CRM1, BARD1, and Aurora A have distinct roles in targeting BRCA1 to the centrosome and regulating its activity. The BRCA1-BARD1 heterodimer binds and ubiquitinates ␥-tubulin to inhibit centrosome amplification and promote microtubule nucleation; regulation of BRCA1 targeting and function at the centrosome is poorly understood. Cancer mutations of the BRCT domains, highly folded protein interaction sequences that mediate binding to phosphoserine peptides [9], are known to impair BRCA1 transcription activity [10], DNA repair function [11], and its ability to suppress tumor formation in mice [12]. CRM1 could potentially provide a docking site for NEScontaining regulators of the centrosome Some evidence for this was shown for nucleophosmin (NPM1), a nucleolar protein whose localization at the centrosome was dependent on binding to CRM1 during mitosis and required to prevent more than one round of centrosome duplication during a cell cycle [33, 34]. We have performed the first systematic mapping of sequences critical for centrosomal localization of BRCA1 and describe evidence supporting distinct roles for the N-terminal binding partners CRM1 and BARD1 and C-terminal binding partner Aurora A in regulating BRCA1 localization, dynamics, and function at the centrosome
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