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A cancer-associated BRCA2 mutation reveals masked nuclear export signals controlling localization

Nature Structural & Molecular Biology volume 20, pages 1191–1198 (2013)Cite this article

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Abstract

Germline missense mutations affecting a single BRCA2 allele predispose humans to cancer. Here we identify a protein-targeting mechanism that is disrupted by the cancer-associated mutation, BRCA2D2723H, and that controls the nuclear localization of BRCA2 and its cargo, the recombination enzyme RAD51. A nuclear export signal (NES) in BRCA2 is masked by its interaction with a partner protein, DSS1, such that point mutations impairing BRCA2-DSS1 binding render BRCA2 cytoplasmic. In turn, cytoplasmic mislocalization of mutant BRCA2 inhibits the nuclear retention of RAD51 by exposing a similar NES in RAD51 that is usually obscured by the BRCA2-RAD51 interaction. Thus, a series of NES-masking interactions localizes BRCA2 and RAD51 in the nucleus. Notably, BRCA2D2723H decreases RAD51 nuclear retention even when wild-type BRCA2 is also present. Our findings suggest a mechanism for the regulation of the nucleocytoplasmic distribution of BRCA2 and RAD51 and its impairment by a heterozygous disease-associated mutation.

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Figure 1: DSS1 binding correlates with localization of BRCA2.
Figure 2: DSS1 regulates a functional NES in BRCA2.
Figure 3: BRCA2 regulates a functional NES in RAD51.
Figure 4: CRM1 binding to NESs in BRCA2 or RAD51 is masked by DSS1 or BRC4, respectively.
Figure 5: RAD51 mislocalization by BRCA2 depletion or mutation.
Figure 6: RAD51 nuclear enrichment is a BRCA2-dependent DNA-damage response.
Figure 7: A hypothetical model for BRCA2 and RAD51 nuclear localization through masking of NESs.

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Acknowledgements

We acknowledge the generous gifts of WT and BRCA2D2723H ES cells from S. Sharan (US National Institutes of Health) and sYFP cDNA from T. Gadella (University of Amsterdam). We thank D. Görlich (Max Planck Institute) for generously providing constructs encoding RanQ69L and CRM1 and for information concerning assay conditions. We thank members of the Venkitaraman laboratory for technical assistance and constructive discussions and M. Goode for help with the feeder-free culture for ES cell cultures. A.D.J. was supported by a career development fellowship from the UK MRC Cancer Unit. Work in the laboratory of C.F.K. was funded by the UK Biosciences and Biotechnology Research Council, and work in the laboratory of A.R.V. was funded by the UK MRC.

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  1. Hiroyoshi Hattori and Venkat Pisupati: These authors contributed equally to this work.

Authors and Affiliations

  1. The Medical Research Council (MRC) Cancer Unit, University of Cambridge, Hutchison–MRC Research Centre, Cambridge, UK

    Anand D Jeyasekharan, Yang Liu, Hiroyoshi Hattori, Venkat Pisupati, Asta Bjork Jonsdottir, Eeson Rajendra, Miyoung Lee, Elayanambi Sundaramoorthy, Yaara Ofir-Rosenfeld, Ko Sato, Jane Savill, Nabieh Ayoub & Ashok R Venkitaraman

  2. Department of Chemical Engineering, University of Cambridge, Cambridge, UK

    Simon Schlachter & Clemens F Kaminski

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Contributions

A.D.J. and A.R.V. conceived the project. Experiments were performed by A.D.J., A.B.J., J.S. and M.L. (microscopy); Y.L. and M.L. (biochemistry); and A.D.J., H.H., V.P., A.B.J., E.S., Y.R., K.S. and N.A. (cell biology). Structural analysis was performed by E.R., and FRET-FLIM analysis was performed by S.S. and C.F.K. A.D.J. and A.R.V. wrote the manuscript.

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Correspondence to Ashok R Venkitaraman.

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Jeyasekharan, A., Liu, Y., Hattori, H. et al. A cancer-associated BRCA2 mutation reveals masked nuclear export signals controlling localization. Nat Struct Mol Biol 20, 1191–1198 (2013). https://doi.org/10.1038/nsmb.2666

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