The BRCA1/BARD1 heterodimer modulates Ran-dependent mitotic spindle assembly

Harvard University, Cambridge, Massachusetts, United States
Cell (Impact Factor: 32.24). 12/2006; 127(3):539-52. DOI: 10.1016/j.cell.2006.08.053
Source: PubMed


The heterodimeric tumor-suppressor complex BRCA1/BARD1 exhibits E3 ubiquitin ligase activity and participates in cell proliferation and chromosome stability control by incompletely defined mechanisms. Here we show that, in both mammalian cells and Xenopus egg extracts, BRCA1/BARD1 is required for mitotic spindle-pole assembly and for accumulation of TPX2, a major spindle organizer and Ran target, on spindle poles. This function is centrosome independent, operates downstream of Ran GTPase, and depends upon BRCA1/BARD1 E3 ubiquitin ligase activity. Xenopus BRCA1/BARD1 forms endogenous complexes with three spindle-pole proteins, TPX2, NuMA, and XRHAMM--a known TPX2 partner--and specifically attenuates XRHAMM function. These observations reveal a previously unrecognized function of BRCA1/BARD1 in mitotic spindle assembly that likely contributes to its role in chromosome stability control and tumor suppression.

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Available from: Vladimir Joukov, Dec 18, 2013
    • "Coordinated transcriptional and post-transcriptional mechanisms ensure mitotic progression in a unidirectional manner by the targeted proteolysis of hundreds of proteins, via ubiquitin–proteasome system or microRNA-mediated translational control, and by time-dependent post-translational modifications, most notably phosphorylation or hydroxylation; such mechanisms lead to the establishment of a specific array of interacting proteins for each cell cycle phase and for the transition between phases, whereas altered expression of mitotic regulators contributes to the generation of aneuploidy, reviewed in (Reed 2003; Bueno and Malumbres 2011; Zhang et al. 2014; Ploumakis and Coleman 2015). The abilities of tumor suppressor proteins, such as adenomatous polyposis coli (APC), phosphatase and tensin homolog, BRCA1, merlin, centromere-associated protein E (CENP-E), and transformic acidic coiledcoil protein (TACC3), to enter the nucleus and to contribute, through protein-stoichiometric regulation of microtubule dynamics, in spindle integrity and proper capturing of chromosomes have come into focus as a major mechanism of action, disruption of which, including deregulated protein dosage, generates CIN (Kaplan et al. 2001; Putkey et al. 2002; Muranen et al. 2005, 2007; Joukov et al. 2006; Liu et al. 2007; Cai et al. 2009; Fu et al. 2010). These functional properties require the presence of a nuclear localization signal (NLS) in the primary sequence of these proteins and are highly controlled by the post-translational modification of phosphorylation, in particular by PKC; phosphorylation, among other functions, regulates the presentation of the NLS for docking the protein onto the Nuclear Pore Complex as a cargo for nuclear import (Topham et al. 1998; Kondoh et al. 2006; Galli et al. 2011). "
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    ABSTRACT: Neurofibromatosis type-1 (NF-1) is caused by mutations in the tumor suppressor gene NF1; its protein product neurofibromin is a RasGAP, a property that has yet to explain aneuploidy, most often observed in astrocytes in NF-1. Here we provide a mechanistic model for the regulated nuclear import of neurofibromin during the cell cycle and for a role in chromosome congression. Specifically, we demonstrate that neurofibromin, phosphorylated on Ser2808, a residue adjacent to an NLS in the C-terminal domain (CTD), by PKC-ε, accumulates in a Ran-dependent manner and through binding to lamin in the nucleus at G2 in glioblastoma cells. Furthermore, we identify CTD as a tubulin-binding domain and show that a phosphomimetic substitution of its Ser2808 results in a predominantly nuclear localization. Confocal analysis shows that endogenous neurofibromin localizes on the centrosomes at interphase, as well as on the mitotic spindle, through direct associations with tubulins, in glioblastoma cells and primary astrocytes. More importantly, analysis of mitotic phenotypes after siRNA-mediated depletion shows that acute loss of this tumor suppressor protein leads to aberrant chromosome congression at the metaphase plate. Therefore, neurofibromin protein abundance and nuclear import are mechanistically linked to an error-free chromosome congression. This article is protected by copyright. All rights reserved.
    No preview · Article · Oct 2015 · Journal of Neurochemistry
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    • "Addition of linear DNA fragments to Xenopus egg extracts mimics the effects of double strand breaks in genomic DNA, enabling study of DNA damage checkpoints in a cell-free model (Willis et al., 2012; You et al., 2007). Detailed methods are also described for studying mitotic spindle assembly and checkpoints (Desai et al., 1999), and many mitotic spindle factors that are altered in cancer are conserved in Xenopus (Cross and Powers, 2009; Joukov et al., 2006). However, Xenopus is not limited to in vitro investigation of cell cycle function; the developing Xenopus embryo also presents an interesting in vivo system to study regulation of proliferation (Woodland, 1974) particularly in view of the changes in the cell cycle regulation during early development (Saka and Smith, 2001). "
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    ABSTRACT: One of the most striking features of the Xenopus system is the versatility in providing a unique range of both in vitro and in vivo models that are rapid, accessible and easily manipulated. Here we present an overview of the diverse contribution that Xenopus has made to advance our understanding of tumour biology and behaviour; a contribution that goes beyond the traditional view of Xenopus as a developmental model organism. From the utility of the egg and oocyte extract system to the use of whole embryos as developmental or induced tumour models, the Xenopus system has been fundamental to investigation of cell cycle mechanisms, cell metabolism, cell signalling and cell behaviour, and has allowed an increasing appreciation of the parallels between early development and the pathogenesis of tumour progression and metastasis. Although not the prototypical oncological model system, we propose that Xenopus is an adaptable and multifunctional tool in the oncologist's arsenal. Copyright © 2015. Published by Elsevier Inc.
    Full-text · Article · Feb 2015 · Developmental Biology
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    • "The X.l.BRCA1-BARD1 heterodimeric complex was purified from insect cells as in Joukov et al. (2006). Full-length FLAG-BRCA1 and HA-BARD1 were each cloned into pFastBac and used to generate the corresponding recombinant baculoviruses (Bac-to-Bac Baculovirus Expression System, Life Technologies, Carlsbad). "
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    ABSTRACT: The tumor suppressor protein BRCA1 promotes homologous recombination (HR), a high-fidelity mechanism to repair DNA double-strand breaks (DSBs) that arise during normal replication and in response to DNA-damaging agents. Recent genetic experiments indicate that BRCA1 also performs an HR-independent function during the repair of DNA interstrand crosslinks (ICLs). Here we show that BRCA1 is required to unload the CMG helicase complex from chromatin after replication forks collide with an ICL. Eviction of the stalled helicase allows leading strands to be extended toward the ICL, followed by endonucleolytic processing of the crosslink, lesion bypass, and DSB repair. Our results identify BRCA1-dependent helicase unloading as a critical, early event in ICL repair.
    Full-text · Article · Sep 2014 · Molecular Cell
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