The nuclear localization sequences of the BRCA1 protein interact with the importin-α subunit of the nuclear transport signal receptor

Chi Fen Chen, Shang Li, Yumay Chen, Phang Lang Chen, Zelton D Sharp, Wen Hwa Lee

Research output: Contribution to journalArticle

173 Citations (Scopus)

Abstract

The BRCA1 gene product is a nuclear phosphoprotein that is aberrantly localized in the cytoplasm of most breast cancer cells. In an attempt to elucidate the potential mechanism for the nuclear transport of BRCA1 protein, three regions of highly charged, basic residues, 503KRKRRP508, 606PKKNRLRRKS615, and 651KKKKYN656, were identified as potential nuclear localization signals (NLSs). These three regions were subsequently mutated to 503KLP508, 607KLS615, and 651KLN656, respectively. Wild-type and mutated proteins were tagged with the flag epitope, expressed in human DU145 cells, and detected with the M2 monoclonal antibody. In DU145 cells, the KLP mutant completely fails to localize in nuclei, whereas the KLS mutant is mostly cytoplasmic with occasional nuclear localization. The KLN protein is always located in nuclei. Consistently, hSRP1α (importin-α), a component of the NLS receptor complex, was identified in a yeast two-hybrid screen using BRCA1 as the bait. The specificity of the interaction between BRCA1 and importin-α was further demonstrated by showing that the 503KRKRRP508 and 606PKKNRLRRKS615 regions, but not 651KKKKYN656, are critical for this interaction. To determine if the cytoplasmic mislocation of endogenous BRCA1 in breast cancer cells is due to a deficiency of the cells, wild-type BRCA1 protein tagged with the flag epitope was ectopically expressed in six breast cancer cell lines. The analysis demonstrated that, in all six, this protein localized in the cytoplasm of these cells. In contrast, expression of the construct in four non-breast cancer cell lines resulted in nuclear localization. These data support the possibility that the mislocation of the BRCA1 protein in breast cancer cells may be due to a defect in the cellular machinery involved in the NLS receptor-mediated pathway of nuclear import.

Original languageEnglish (US)
Pages (from-to)32863-32868
Number of pages6
JournalJournal of Biological Chemistry
Volume271
Issue number51
DOIs
StatePublished - 1996

Fingerprint

BRCA1 Protein
Karyopherins
Cell Nucleus Active Transport
Cells
Nuclear Localization Signals
Breast Neoplasms
Epitopes
Proteins
Cytoplasm
Phosphoproteins
Yeast
Cell Line
Machinery
Monoclonal Antibodies
Nuclear Proteins
Defects
Carrier Proteins
Yeasts

ASJC Scopus subject areas

  • Biochemistry

Cite this

The nuclear localization sequences of the BRCA1 protein interact with the importin-α subunit of the nuclear transport signal receptor. / Chen, Chi Fen; Li, Shang; Chen, Yumay; Chen, Phang Lang; Sharp, Zelton D; Lee, Wen Hwa.

In: Journal of Biological Chemistry, Vol. 271, No. 51, 1996, p. 32863-32868.

Research output: Contribution to journalArticle

Chen, Chi Fen ; Li, Shang ; Chen, Yumay ; Chen, Phang Lang ; Sharp, Zelton D ; Lee, Wen Hwa. / The nuclear localization sequences of the BRCA1 protein interact with the importin-α subunit of the nuclear transport signal receptor. In: Journal of Biological Chemistry. 1996 ; Vol. 271, No. 51. pp. 32863-32868.
@article{815b18cfddd34a55ab7d638cddb9cbac,
title = "The nuclear localization sequences of the BRCA1 protein interact with the importin-α subunit of the nuclear transport signal receptor",
abstract = "The BRCA1 gene product is a nuclear phosphoprotein that is aberrantly localized in the cytoplasm of most breast cancer cells. In an attempt to elucidate the potential mechanism for the nuclear transport of BRCA1 protein, three regions of highly charged, basic residues, 503KRKRRP508, 606PKKNRLRRKS615, and 651KKKKYN656, were identified as potential nuclear localization signals (NLSs). These three regions were subsequently mutated to 503KLP508, 607KLS615, and 651KLN656, respectively. Wild-type and mutated proteins were tagged with the flag epitope, expressed in human DU145 cells, and detected with the M2 monoclonal antibody. In DU145 cells, the KLP mutant completely fails to localize in nuclei, whereas the KLS mutant is mostly cytoplasmic with occasional nuclear localization. The KLN protein is always located in nuclei. Consistently, hSRP1α (importin-α), a component of the NLS receptor complex, was identified in a yeast two-hybrid screen using BRCA1 as the bait. The specificity of the interaction between BRCA1 and importin-α was further demonstrated by showing that the 503KRKRRP508 and 606PKKNRLRRKS615 regions, but not 651KKKKYN656, are critical for this interaction. To determine if the cytoplasmic mislocation of endogenous BRCA1 in breast cancer cells is due to a deficiency of the cells, wild-type BRCA1 protein tagged with the flag epitope was ectopically expressed in six breast cancer cell lines. The analysis demonstrated that, in all six, this protein localized in the cytoplasm of these cells. In contrast, expression of the construct in four non-breast cancer cell lines resulted in nuclear localization. These data support the possibility that the mislocation of the BRCA1 protein in breast cancer cells may be due to a defect in the cellular machinery involved in the NLS receptor-mediated pathway of nuclear import.",
author = "Chen, {Chi Fen} and Shang Li and Yumay Chen and Chen, {Phang Lang} and Sharp, {Zelton D} and Lee, {Wen Hwa}",
year = "1996",
doi = "10.1074/jbc.271.51.32863",
language = "English (US)",
volume = "271",
pages = "32863--32868",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "51",

}

TY - JOUR

T1 - The nuclear localization sequences of the BRCA1 protein interact with the importin-α subunit of the nuclear transport signal receptor

AU - Chen, Chi Fen

AU - Li, Shang

AU - Chen, Yumay

AU - Chen, Phang Lang

AU - Sharp, Zelton D

AU - Lee, Wen Hwa

PY - 1996

Y1 - 1996

N2 - The BRCA1 gene product is a nuclear phosphoprotein that is aberrantly localized in the cytoplasm of most breast cancer cells. In an attempt to elucidate the potential mechanism for the nuclear transport of BRCA1 protein, three regions of highly charged, basic residues, 503KRKRRP508, 606PKKNRLRRKS615, and 651KKKKYN656, were identified as potential nuclear localization signals (NLSs). These three regions were subsequently mutated to 503KLP508, 607KLS615, and 651KLN656, respectively. Wild-type and mutated proteins were tagged with the flag epitope, expressed in human DU145 cells, and detected with the M2 monoclonal antibody. In DU145 cells, the KLP mutant completely fails to localize in nuclei, whereas the KLS mutant is mostly cytoplasmic with occasional nuclear localization. The KLN protein is always located in nuclei. Consistently, hSRP1α (importin-α), a component of the NLS receptor complex, was identified in a yeast two-hybrid screen using BRCA1 as the bait. The specificity of the interaction between BRCA1 and importin-α was further demonstrated by showing that the 503KRKRRP508 and 606PKKNRLRRKS615 regions, but not 651KKKKYN656, are critical for this interaction. To determine if the cytoplasmic mislocation of endogenous BRCA1 in breast cancer cells is due to a deficiency of the cells, wild-type BRCA1 protein tagged with the flag epitope was ectopically expressed in six breast cancer cell lines. The analysis demonstrated that, in all six, this protein localized in the cytoplasm of these cells. In contrast, expression of the construct in four non-breast cancer cell lines resulted in nuclear localization. These data support the possibility that the mislocation of the BRCA1 protein in breast cancer cells may be due to a defect in the cellular machinery involved in the NLS receptor-mediated pathway of nuclear import.

AB - The BRCA1 gene product is a nuclear phosphoprotein that is aberrantly localized in the cytoplasm of most breast cancer cells. In an attempt to elucidate the potential mechanism for the nuclear transport of BRCA1 protein, three regions of highly charged, basic residues, 503KRKRRP508, 606PKKNRLRRKS615, and 651KKKKYN656, were identified as potential nuclear localization signals (NLSs). These three regions were subsequently mutated to 503KLP508, 607KLS615, and 651KLN656, respectively. Wild-type and mutated proteins were tagged with the flag epitope, expressed in human DU145 cells, and detected with the M2 monoclonal antibody. In DU145 cells, the KLP mutant completely fails to localize in nuclei, whereas the KLS mutant is mostly cytoplasmic with occasional nuclear localization. The KLN protein is always located in nuclei. Consistently, hSRP1α (importin-α), a component of the NLS receptor complex, was identified in a yeast two-hybrid screen using BRCA1 as the bait. The specificity of the interaction between BRCA1 and importin-α was further demonstrated by showing that the 503KRKRRP508 and 606PKKNRLRRKS615 regions, but not 651KKKKYN656, are critical for this interaction. To determine if the cytoplasmic mislocation of endogenous BRCA1 in breast cancer cells is due to a deficiency of the cells, wild-type BRCA1 protein tagged with the flag epitope was ectopically expressed in six breast cancer cell lines. The analysis demonstrated that, in all six, this protein localized in the cytoplasm of these cells. In contrast, expression of the construct in four non-breast cancer cell lines resulted in nuclear localization. These data support the possibility that the mislocation of the BRCA1 protein in breast cancer cells may be due to a defect in the cellular machinery involved in the NLS receptor-mediated pathway of nuclear import.

UR - http://www.scopus.com/inward/record.url?scp=12644270610&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=12644270610&partnerID=8YFLogxK

U2 - 10.1074/jbc.271.51.32863

DO - 10.1074/jbc.271.51.32863

M3 - Article

C2 - 8955125

AN - SCOPUS:12644270610

VL - 271

SP - 32863

EP - 32868

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 51

ER -