Skip to main content

Identification of a non-canonical nuclear localization signal (NLS) in BRCA1 that could mediate nuclear localization of splice variants lacking the classical NLS

Abstract

The breast cancer type 1 susceptibility gene (BRCA1) is a tumor suppressor gene, mutations or loss of which lead to genomic instability and breast cancer. BRCA1 protein is part of a large multi-protein complex involved in a variety of DNA repair and transcription regulatory functions. At least four splice variants have been described and these differ in their function and tissue and spatio-temporal expression patterns. Structural analysis has revealed the presence of two nuclear localization signals (NLS) located in exon 11 of BRCA1. Interestingly, a splice variant of the protein that lacks both of the known NLS still manages to gain entry to the nucleus. While there is experimental proof for the translocation of these proteins by binding to other established nuclear proteins, we examined the possibility of a hitherto unidentified NLS in this particular variant. In this paper, we present evidence for the existence of a previously unreported non-canonical NLS contained within the first 39 amino acids of exon 11. A fusion protein with this 39mer and a reporter green fluorescent protein translocated into the nucleus when it was expressed in breast epithelial cells. We demonstrate the presence of a hitherto unreported noncanonical NLS in exon 11a of BRCA1. This NLS might aid proteins that were encoded by splice variants and lack the canonical NLS to localize to the nucleus.

Abbreviations

BARD1:

BRCA1-associated RING domain

BRCA1 :

breast cancer type 1 susceptibility protein

DAPI:

4′,6-diamidino-2-phenylindole

EGFP:

enhanced green fluorescent protein

GFP:

green fluorescent protein

HBL-100:

human breast carcinoma cell line

HEK-293:

human embryonic kidney 293 cells

NLS:

nuclear localization signals

Ubc9:

ubiquitin-conjugating enzyme 9

References

  1. Miki, Y., Swensen, J., Shattuck-Eidens, D., Futreal, P.A., Harshman, K., Tavtigian, S., Liu, Q., Cochran, C., Bennett, L.M. and Ding, W. A strong candidate for the breast and ovarian cancer susceptibility gene BRCA1. Science 266 (1994) 66–71.

    PubMed  Article  CAS  Google Scholar 

  2. Rosen, E.M., Fan, S., Pestell, R.G. and Goldberg, I.D. BRCA1 gene in breast cancer. J. Cell. Physiol. 196 (2003) 19–41.

    PubMed  Article  CAS  Google Scholar 

  3. Rakha, E.A., El-Sheikh, S.E., Kandil, M.A., El-Sayed, M.E., Green, A.R. and Ellis, I.O. Expression of BRCA1 protein in breast cancer and its prognostic significance. Hum. Pathol. 39 (2008) 857–865.

    PubMed  Article  CAS  Google Scholar 

  4. Mullan, P.B., Quinn, J.E. and Harkin, D.P. The role of BRCA1 in transcriptional regulation and cell cycle control. Oncogene 25 (2006) 5854–5863.

    PubMed  Article  CAS  Google Scholar 

  5. Monteiro, A.N., August, A. and Hanafusa, H. Evidence for a transcriptional activation function of BRCA1 C-terminal region. Proc. Natl. Acad. Sci. USA 93 (1996)13595–13599.

    PubMed  Article  CAS  Google Scholar 

  6. Boulton, S.J. BRCA1-mediated ubiquitylation. Cell Cycle 5 (2006) 1481–1486.

    PubMed  Article  CAS  Google Scholar 

  7. Thakur, S., Zhang, H.B., Peng, Y., Le, H., Carroll, B., Ward, T., Yao, J., Farid, L.M., Couch, F.J. and Wilson, R.B. Localization of BRCA1 and a splice variant identifies the nuclear localization signal. Mol. Cell. Biol. 17 (1997) 444–452.

    PubMed  CAS  Google Scholar 

  8. Chen, C.F., Li, S., Chen, Y., Chen, P.L., Sharp, Z.D. and Lee, W.H. The nuclear localization sequences of the BRCA1 protein interact with the importin-alpha subunit of the nuclear transport signal receptor. J. Biol. Chem. 271 (1996) 32863–32868.

    PubMed  Article  CAS  Google Scholar 

  9. Orban, T.I. and Olah, E. Expression profiles of BRCA1 splice variants in asynchronous and in G1/S synchronized tumor cell lines. Bioch. Biophys. Res. Commun. 280 (2001) 32–38.

    Article  CAS  Google Scholar 

  10. Wang, H., Shao, N., Ding, Q.M., Cui, J., Reddy, E.S. and Rao, V.N. BRCA1 proteins are transported to the nucleus in the absence of serum and splice variants BRCA1a, BRCA1b are tyrosine phosphoproteins that associate with E2F, cyclins and cyclin dependent kinases. Oncogene 15 (1997)143–157.

    PubMed  Article  CAS  Google Scholar 

  11. Subedi, K.P., Singh, T.D., Kim, J.C. and Woo, S.H. Cloning and expression of a new inositol 1,4,5-trisphosphate receptor type 1 splice variant in adult rat atrial myocytes. Cell Mol. Biol. Lett. 17 (2012) 124–135.

    PubMed  Article  CAS  Google Scholar 

  12. Wilson, C.A., Payton, M.N., Elliott, G.S., Buaas, F.W., Cajulis, E.E., Grosshans, D., Ramos, L., Reese, D.M., Slamon, D.J. and Calzone, F.J. Differential subcellular localization, expression and biological toxicity of BRCA1 and the splice variant BRCA1-delta11b. Oncogene 14 (1997) 1–16.

    PubMed  Article  CAS  Google Scholar 

  13. Maniccia, A.W., Lewis, C., Begum, N., Xu, J., Cui, J., Chipitsyna, G., Aysola, K., Reddy, V., Bhat, G., Fujimura, Y., Henderson, B., Reddy, E.S. and Rao, V.N. Mitochondrial localization, ELK-1 transcriptional regulation and growth inhibitory functions of BRCA1, BRCA1a, and BRCA1b proteins. J. Cell Physiol. 219 (2009) 634–641.

    PubMed  Article  CAS  Google Scholar 

  14. Cui, J.Q., Shao, N., Chai, Y., Wang, H., Reddy, E.S. and Rao, V.N. BRCA1 splice variants BRCA1a and BRCA1b associate with CBP co-activator. Oncol. Rep. 5 (1998) 591–595.

    PubMed  CAS  Google Scholar 

  15. Qin, Y., Xu, J., Aysola, K., Begum, N., Reddy, V., Chai, Y., Grizzle, W.E., Partridge, E.E., Reddy, E.S. and Rao, V.N. Ubc9 mediates nuclear localization and growth suppression of BRCA1 and BRCA1a proteins. J. Cell Physiol. 226 (2011) 3355–3367.

    PubMed  Article  CAS  Google Scholar 

  16. Xu, J., Watkins, T., Reddy, A., Reddy, E.S. and Rao, V.N. A novel mechanism whereby BRCA1/1a/1b fine tunes the dynamic complex interplay between SUMO-dependent/independent activities of Ubc9 on E2-induced ERalpha activation/repression and degradation in breast cancer cells. Int. J. Oncology 34 (2009) 939–949.

    CAS  Google Scholar 

  17. Fabbro, M., Rodriguez, J.A., Baer, R. and Henderson, B.R. BARD1 induces BRCA1 intranuclear foci formation by increasing RING-dependent BRCA1 nuclear import and inhibiting BRCA1 nuclear export. J. Biol. Chem. 277 (2002) 21315–21324.

    PubMed  Article  CAS  Google Scholar 

  18. Grunwald, M. and Bono, F. Structure of Importin13-Ubc9 complex: nuclear import and release of a key regulator of sumoylation. EMBO J. 30 (2011) 427–438.

    PubMed  Article  Google Scholar 

  19. Wente, S.R. and Rout, M.P. The nuclear pore complex and nuclear transport. Cold Spring Harb. Perspect. Biol. 2 (2010) 14.

    Article  Google Scholar 

  20. Kalderon, D., Roberts, B.L., Richardson, W.D. and Smith, A.E. A short amino acid sequence able to specify nuclear location. Cell 39 (1984) 499–509.

    PubMed  Article  CAS  Google Scholar 

  21. Lange, A., Mills, R.E., Lange, C.J., Stewart, M., Devine, S.E. and Corbett, A.H. Classical nuclear localization signals: definition, function, and interaction with importin alpha. J. Biol. Chem. 282 (2007) 5101–5105.

    PubMed  Article  CAS  Google Scholar 

  22. Schwemmle, M., Jehle, C., Shoemaker, T. and Lipkin, W.I. Characterization of the major nuclear localization signal of the Borna disease virus phosphoprotein. J. Gen. Virol. 80 (1999) 97–100.

    PubMed  CAS  Google Scholar 

  23. Seibel, N.M., Eljouni, J., Nalaskowski, M.M. and Hampe, W. Nuclear localization of enhanced green fluorescent protein homomultimers. Anal. Biochem. 368 (2007) 95–99.

    PubMed  Article  CAS  Google Scholar 

  24. Dross, N., Spriet, C., Zwerger, M., Muller, G., Waldeck, W. and Langowski, J. Mapping eGFP oligomer mobility in living cell nuclei. PLoS One 4 (2009) 4.

    Article  Google Scholar 

  25. Morinelli, T.A., Raymond, J.R., Baldys, A., Yang, Q., Lee, M.H., Luttrell, L. and Ullian, M.E. Identification of a putative nuclear localization sequence within ANG II AT(1A) receptor associated with nuclear activation. Am. J. Physiol. Cell. Physiol. 292 (2007) 13.

    Google Scholar 

  26. Lyssenko, N.N., Hanna-Rose, W. and Schlegel, R.A Cognate putative nuclear localization signal effects strong nuclear localization of a GFP reporter and facilitates gene expression studies in Caenorhabditis elegans. Biotechniques 43 (2007) 560.

    Article  Google Scholar 

  27. Minopoli, G., de Candia, P., Bonetti, A., Faraonio, R., Zambrano, N. and Russo, T. The beta-amyloid precursor protein functions as a cytosolic anchoring site that prevents Fe65 nuclear translocation. J. Biol. Chem. 276 (2001) 6545–6550.

    PubMed  Article  CAS  Google Scholar 

  28. Borghi, S., Molinari, S., Razzini, G., Parise, F., Battini, R. and Ferrari, S. The nuclear localization domain of the MEF2 family of transcription factors shows member-specific features and mediates the nuclear import of histone deacetylase 4. J. Cell Sci. 114 (2001) 4477–4483.

    PubMed  CAS  Google Scholar 

  29. Von Kobbe, C. and Bohr, V.A. A nucleolar targeting sequence in the Werner syndrome protein resides within residues 949–1092. J. Cell Sci. 115 (2002) 3901–3907.

    Article  Google Scholar 

  30. Henderson, B.R. Regulation of BRCA1, BRCA2 and BARD1 intracellular trafficking. BioEssays 27 (2005) 884–893.

    PubMed  Article  CAS  Google Scholar 

  31. Turner, N., Tutt, A. and Ashworth, A. Hallmarks of ‘BRCAness’ in sporadic cancers. Nat. Rev. Cancer 4 (2004) 814–819.

    PubMed  Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Aruna Korlimarla.

Additional information

These authors contributed equally to this manuscript

Visiting Scientist, SJRI

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Korlimarla, A., Bhandary, L., Prabhu, J.S. et al. Identification of a non-canonical nuclear localization signal (NLS) in BRCA1 that could mediate nuclear localization of splice variants lacking the classical NLS. Cell Mol Biol Lett 18, 284–296 (2013). https://doi.org/10.2478/s11658-013-0088-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.2478/s11658-013-0088-x

Key words

  • Breast cancer
  • BRCA1
  • Splice variants
  • Nuclear localization
  • Importin alpha
  • Green fluorescent protein