Nuclear targeting and cell cycle regulatory function of human BARD1.
The BARD1 gene is mutated in a subset of breast and ovarian cancers, implicating BARD1 as a potential tumor suppressor. BARD1 gains a ubiquitin E3 ligase activity when heterodimerized with BRCA1, but the only known BRCA1-independent BARD1 function is a p53-dependent proapoptotic activity stimulated by nuclear export to the cytoplasm. We described previously the nuclear-cytoplasmic shuttling of BARD1, and in this study, we identify the transport sequences that target BARD1 to the nucleus and show that they are essential for BARD1 regulation of the cell cycle. We used deletion mapping and mutagenesis to define two active nuclear localization signals (NLSs) present in human BARD1 that are not conserved in rodent BARD1. Site-directed mutagenesis of the primary bipartite NLS abolished BARD1 nuclear import and caused its cytoplasmic accumulation. Using flow cytometry and 5-bromo-2-deoxyuridine incorporation assays, we discovered that transiently expressed BARD1 can elicit a p53-independent cell cycle arrest in G1 phase, and that this was abrogated by mutation of the BARD1 NLS but not by mutation of the nuclear export signal. Thus, BARD1 regulation of the cell cycle is a nuclear event and may be linked to its induced expression during mitosis and its possible involvement in the DNA damage checkpoint.
Schüchner
Stefan
S
Westmead Institute for Cancer Research, University of Sydney, Westmead Millennium Institute at Westmead Hospital, Darcy Road, PO Box 412, Westmead, Sydney, New South Wales 2145, Australia.
Tembe
Varsha
V
Rodriguez
José A
JA
Henderson
Beric R
BR
D000595:Amino Acid Sequence; D019313:BRCA1 Protein / Q000378:metabolism; D001943:Breast Neoplasms; D002453:Cell Cycle / Q000502:physiology*; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000502:physiology*; D005260:Female; D016193:G1 Phase / Q000502:physiology; D006706:Homeostasis; D006801:Humans; D008938:Mitosis; D008969:Molecular Sequence Data; D021381:Protein Transport; D017384:Sequence Deletion; D014162:Transfection; D025521:Tumor Suppressor Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016335:Zinc Fingers
D019313:BRCA1 Protein; D025521:Tumor Suppressor Proteins; C102449:BARD1 protein, human; D044767:Ubiquitin-Protein Ligases