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 Huichen Wang 1,3 , Ningsheng Shao 1,3 , Qing Ming Ding 1,3 , Jian-qi Cui 1 , E Shyam P Reddy 1 and Veena N Rao 1,2 1 Division of Cancer Genetics, Department of Human Genetics, Allegheny University of the Health Sciences, M.S. 481, New College Building, Broad and Vine Streets, Philadelphia, Pennsylvania 19102, USA BRCA1, a familial breast and ovarian cancer suscept- ibility gene encodes nuclear phosphoproteins that func- tion as tumor suppressors in human breast cancer cells. Previously, we have shown that overexpression of a BRCA1 splice variant BRCA1a accelerates apoptosis in human breast cancer cells. In an attempt to determine whether the subcellular localization of BRCA1 is cell cycle regulated, we have studied the subcellular distribu- tion of BRCA1 in asynchronous and growth arrested normal, breast and ovarian cancer cells using dierent BRCA1 antibodies by immuno¯uorescence and immuno- histochemical staining. Upon serum starvation of NIH3T3, some breast and ovarian cancer cells, most of the BRCA1 protein redistributed to the nucleus revealing a new type of regulation that may modulate the activity of BRCA1 gene. We have also characterized two new variant BRCA1 proteins (BRCA1a/p110 and BRCA1b/ p100) which are phosphoproteins containing phosphotyr- osine. Immuno¯uorescence and Western blotting analysis indicate cytoplasmic and nuclear localization of BRCA1a and BRCA1b proteins. To elucidate the biological function of BRCA1, we created a bacterial fusion protein of glutathione-transferase (GST) and BRCA1 zinc ®nger domain and detected two cellular proteins with molecular weights of approximately 32 and 65 kD, one of which contains phosphotyrosine designated p32 and p65 BRCA1 interacting proteins (BIP) that speci®cally interact with BRCA1. Western blot analysis of BIP with cyclins/CDKs and E2F antisera indicated association with cdc2, cdk2, cdk4, cyclin B, cyclin D, cyclin A and E2F-4 but not with cdk3, cdk5, cdk6, E2F- 1, E2F-2, E2F-3, E2F-5 and cyclin E. Furthermore, we have also demonstrated a direct interaction of in vitro translated BRCA1a and BRCA1b proteins with recom- binant cyclin A, cyclin B1, cyclin D1, cdc2, cdk2 and E2F fusion proteins in vitro. Taken together these results seem to suggest that BRCA1 could be an important negative regulator of cell cycle that functions through interaction with E2F transcriptional factors and phos- phorylation by cyclins/cdk complexes with the zinc ring ®nger functioning as a major protein-protein interaction domain. If the interactions we observe in vitro is also seen in vivo then it may be possible that lack or impaired binding of the disrupted BRCA1 proteins to E2F, cyclins/CDKs in patients with mutations in the zinc ®nger domain could deprive the cell of an important mechanism for braking cell proliferation leading to the development of breast and ovarian cancers. Keywords: BRCA1a; BRCA1b; zinc ®nger; cyclins; CDKs; E2F Introduction Mutations in the breast and ovarian cancer suscept- ibility gene BRCA1, accounts for half of the inherited breast and ovarian cancers (Miki et al., 1994; Easton et al., 1995; Ford et al., 1995) and only 10% of the sporadic ovarian cancers (Futreal et al., 1994; Hosking et al., 1995; Merajver et al., 1995). The BRCA1 cDNA codes for a 1863 amino acid protein with an amino terminal zinc ring ®nger domain and a carboxy terminal acidic region (Miki et al., 1994) typical of several transcriptional factors. Recently, the C terminal region of BRCA1 was shown to activate transcription in a heterologous GAL-4 system (Chapman and Verma, 1996; Monteiro, 1996; Rao et al., unpublished results). Several groups have cloned and studied the developmental patterns of expression of murine BRCA1 (Lane et al., 1995; Marquis et al., 1995; Abel et al., 1995; Sharan et al., 1995). Expression was found to be high in rapidly proliferating tissues (Lane et al., 1995; Marquis et al., 1995) particularly those under- going dierentiation suggesting a role for BRCA1 in cellular growth and dierentiation. We and others have shown the BRCA1 gene product to be a nuclear phosphoprotein (Chen et al., 1995; Rao et al., 1996; Scully et al., 1996), that when over expressed in breast and ovarian cancer cells results in growth inhibition in vitro and in vivo in breast cancer cells (Holt et al., 1996; Rao et al., unpublished results). Conversely, inhibition of BRCA1 expression by antisense RNA in mouse ®broblasts or by antisense oligonucleotides in breast cancer cells resulted in transformation of mouse ®broblasts as well as increased the rate of growth of breast cancer cells (Thompson et al., 1995; Rao et al., 1996). We have recently reported a new function for the BRCA1 splice variant BRCA1a in the regulation of apoptosis of human breast cancer cells (Shao et al., 1996). Previously, the BRCA1 gene product was shown to be localized in the nucleus (Chen et al., 1995; Rao et al., 1996). Since then there have been several Correspondence: VN Rao 2 Part of this work was conducted at the Department of Microbiology and Immunology, Jeerson Medical College, Kimmel Cancer Institute, 233 South 10th Street, Philadelphia, PA 19107 3 These authors contributed equally to this work Received 8 August 1996; revised 2 May 1997; accepted 6 May 1997 Oncogene (1997) 15, 143 ± 157  1997 Stockton Press All rights reserved 0950 ± 9232/97 $12.00