Subcellular localization and analysis of apparent 180-kDa and 220-kDa proteins of the breast cancer susceptibility gene, BRCA1.
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.
Cancer Research, Lilly Research Laboratories, Eli Lilly and Co., Lilly Corporate Center, Indianapolis, Indiana 46285, USA. Thomas_James_E@Lilly.com
J E
JE
Thomas
M
M
Smith
B
B
Rubinfeld
M
M
Gutowski
R P
RP
Beckmann
P
P
Polakis
D000906:Antibodies; D019313:BRCA1 Protein / Q000032:analysis* / Q000737:chemistry; D001943:Breast Neoplasms / Q000737:chemistry; D005260:Female; D005455:Fluorescent Antibody Technique; D019398:Genes, BRCA1; D006801:Humans; D008970:Molecular Weight; D010051:Ovarian Neoplasms / Q000737:chemistry; D013347:Subcellular Fractions / Q000737:chemistry*; D014407:Tumor Cells, Cultured; D002583:Uterine Cervical Neoplasms / Q000737:chemistry
D000906:Antibodies; D019313:BRCA1 Protein