title: Characterization of BARD1 targeting and dynamics at the centrosome: the role of CRM1, BRCA1 and the Q564H mutation.
abstract: 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.
authors: Westmead Institute for Cancer Research, The University of Sydney, Westmead Millennium Institute at Westmead Hospital, Westmead, NSW 2145, Australia.
authors: Kirsty M
authors: KM
authors: Brodie
authors: Westmead Institute for Cancer Research, The University of Sydney, Westmead Millennium Institute at Westmead Hospital, Westmead, NSW 2145, Australia.
authors: Myth T S
authors: MTS
authors: Mok
authors: Westmead Institute for Cancer Research, The University of Sydney, Westmead Millennium Institute at Westmead Hospital, Westmead, NSW 2145, Australia. Electronic address: beric.henderson@sydney.edu.au.
authors: Beric R
authors: BR
authors: Henderson
mesh_terms: D021581:Active Transport, Cell Nucleus / Q000235:genetics / Q000528:radiation effects; D019313:BRCA1 Protein / Q000235:genetics / Q000378:metabolism*; D001943:Breast Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000235:genetics / Q000378:metabolism; D018385:Centrosome / Q000378:metabolism / Q000528:radiation effects; D003110:Colonic Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D004249:DNA Damage / Q000528:radiation effects; D004260:DNA Repair / Q000528:radiation effects; D019281:Dimerization; D005453:Fluorescence; D036681:Fluorescence Recovery After Photobleaching; D005720:Gamma Rays; D006801:Humans; D028884:Karyopherins / Q000235:genetics / Q000378:metabolism*; D008938:Mitosis / Q000528:radiation effects; D009154:Mutation; D010957:Plasmids; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000528:radiation effects; D014162:Transfection; D025521:Tumor Suppressor Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000097606:Exportin 1 Protein
chemical_list: D019313:BRCA1 Protein; D028884:Karyopherins; D018160:Receptors, Cytoplasmic and Nuclear; D025521:Tumor Suppressor Proteins; D044767:Ubiquitin-Protein Ligases; D000097606:Exportin 1 Protein; C492913:BRCA1 protein, human; C102449:BARD1 protein, human