BRCA1 PROTEINS ARE TRANSPORTED TO THE NUCLEUS IN THE ABSENCE OF SERUMAND SPLICE VARIANTS BRCA1A, BRCA1B ARE TYROSINE PHOSPHOPROTEINS THAT ASSOCIATE WITH E2F, CYCLINS AND CYCLIN-DEPENDENT KINASES

BRCA1, a familial breast and ovarian canter susceptibility gene encode s nuclear phosphoproteins that function 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 ca ncer cells, In an attempt to determine whether the subcellular localiz ation of BRCA1 is cell cycle regulated, we have studied the subcellula r distribution of BRCA1 in asynchronous and growth arrested normal, br east and ovarian cancer cells using different BRCA1 antibodies by immu nofluorescence and immunohistochemical staining, Upon serum starvation of NIH3T3, some breast and ovarian cancer cells, most of the BRCA1 pr otein redistributed to the nucleus revealing a new type of regulation that may modulate the activity of BRCA1 gene, We have also characteriz ed two new variant BRCA1 proteins (BRCA1a/p110 and BRCA1b/p100) which are phosphoproteins containing phosphotyrosine, Immunofluorescence and Western blotting analysis indicate cytoplasmic and nuclear localizati on of BRCA1a and BRCA1b proteins. To elucidate the biological function of BRCA1, we created a bacterial fusion protein of glutathione-transf erase (GST) and BRCA1 zinc finger domain and detected two cellular pro teins with molecular weights of approximately 32 and 65 kD, one of whi ch contains phosphotyrosine designated p32 and p65 BRCA1 interacting p roteins (BIP) that specifically interact with BRCA1, Western blot anal ysis of BIP with cyclins/CDKs and E2F antisera indicated association w ith cdc2, cdk2, cdk4, cyclin B, cyclin D, cyclin A and E2F-4 but not w ith cdk3, cdk5, cdk6, E2F-1, E2F-2, E2F-3, E2F-5 and cyclin E, Further more, we have also demonstrated a direct interaction of in vitro trans lated BRCA1a and BRCA1b proteins with recombinant cyclin A, cyclin B1, cyclin D1, cdc2, cdk2 and E2F fusion proteins in vitro, Taken togethe r these results seem to suggest that BRCA1 could be an important negat ive regulator of cell cycle that functions through interaction with E2 F transcriptional factors and phosphorylation by cyclins/cdk complexes with the zinc ring finger functioning as a major protein-protein inte raction domain, If the interactions we observe in vitro is also seen i n vivo then it may be possible that lack or impaired binding of the di srupted BRCA1 proteins to E2F, cyclins/CDKs in patients with mutations in the zinc finger domain could deprive the cell of an important mech anism for braking cell proliferation leading to the development of bre ast and ovarian cancers.