SUBCELLULAR-LOCALIZATION AND ANALYSIS OF APPARENT 180-KDA AND 220-KDAPROTEINS 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 sub cellular location. We have identified a 220-kDa protein, which is reac tive with three antibodies raised against the amino- and carboxy-termi nal 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 ind icated 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 t wo 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 recepto r; (ii) an EGF receptor antibody detected a 180-kDa protein immunoprec ipitated by C-20; (iii) the affinity purified EGF receptor was both im munoprecipitated and detected on immunoblots by the C-20 antibody but not another BRCA1 antibody; (iv) similar phosphopeptide maps were gene rated from the EGF receptor and the 180-kDa protein immunoprecipitated by C-20, and this peptide map was distinct from the 220-kDa phosphopr otein; and (v) the C-20 immunizing peptide bears sequence identity to the EGF receptor. These results indicate that BRCA1 is a 220-kDa nucle ar protein and that the 180-kDa protein reported previously may be unr elated to BRCA1.