Subcellular localization and analysis of apparent 180-kDa and 220-kDa proteins of the breast cancer susceptibility gene, BRCA1

J Biol Chem. 1996 Nov 8;271(45):28630-5. doi: 10.1074/jbc.271.45.28630.

Abstract

The breast cancer susceptibility gene BRCA1 encodes an 1863-amino acid protein that acts as a tumor suppressor. The biochemical function of BRCA1 is unknown, and there are conflicting results describing its subcellular location. We have identified a 220-kDa protein, which is reactive with three antibodies raised against the amino- and carboxyl-terminal regions of BRCA1. Immunoflourescence staining with an antibody to the carboxyl terminus of BRCA1 localized the protein to the nucleus of breast, ovarian, and cervical carcinoma-derived cell lines. A similar result was observed by biochemical subcellular fractionation that indicated that the 220-kDa protein was localized primarily to the nucleus of cell lines established from breast carcinomas. In addition to the 220-kDa protein, one antibody, C-20, also recognized a 180-kDa protein in MDA-MB-468 total cell lysates that was not detected by the other two antibodies. Several observations suggest the 180-kDa protein is the epidermal growth factor (EGF) receptor: (i) C-20 reacted avidly with a 180-kDa protein immunoprecipitated by an antibody to the EGF receptor; (ii) an EGF receptor antibody detected a 180-kDa protein immunoprecipitated by C-20; (iii) the affinity purified EGF receptor was both immunoprecipitated and detected on immunoblots by the C-20 antibody but not another BRCA1 antibody; (iv) similar phosphopeptide maps were generated from the EGF receptor and the 180-kDa protein immunoprecipitated by C-20, and this peptide map was distinct from the 220-kDa phosphoprotein; and (v) the C-20 immunizing peptide bears sequence identity to the EGF receptor. These results indicate that BRCA1 is a 220-kDa nuclear protein and that the 180-kDa protein reported previously may be unrelated to BRCA1.

MeSH terms

  • Antibodies
  • BRCA1 Protein / analysis*
  • BRCA1 Protein / chemistry
  • Breast Neoplasms / chemistry
  • Female
  • Fluorescent Antibody Technique
  • Genes, BRCA1*
  • Humans
  • Molecular Weight
  • Ovarian Neoplasms / chemistry
  • Subcellular Fractions / chemistry*
  • Tumor Cells, Cultured
  • Uterine Cervical Neoplasms / chemistry

Substances

  • Antibodies
  • BRCA1 Protein