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

Abstract

BRCA1, a familial breast and ovarian cancer susceptibility gene encodes 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 cancer cells. In an attempt to determine whether the subcellular localization of BRCA1 is cell cycle regulated, we have studied the subcellular distribution of BRCA1 in asynchronous and growth arrested normal, breast and ovarian cancer cells using dierent BRCA1 antibodies by immuno¯uorescence and immunohistochemical 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 phosphotyrosine. 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 recombinant 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 phosphorylation 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.

Functional consequences of cyclin D1/BRCA1 interaction in breast cancer cells

“…Previous studies have shown that the first 76 aa of BRCA1 interact with multiple cell-cycle factors including cdc2, cdk2, cdk4, cyclin B, cyclin D, cyclin A and E2F-4 (Wang et al, 1997). Our data demonstrates that the principal areas of interaction are the N terminus (aa 1-500) and aa 504-802.…”

“…Likewise, various cyclin/cdk complexes, including cyclin D, have been shown to phosphorylate BRCA1 (Wang et al, 1997;Ruffner et al, 1999); however, the consequence of cyclin D1/cdk phosphorylation on BRCA1 has not been determined.…”

c-Kit is required for growth and survival of the cells of origin of Brca1-mutation-associated breast cancer

“…Lyn is a potential regulator of Brca1, acting via Akt Altiok et al, 1999;Shivakrupa and Linnekin, 2005). Brca1 is itself a potential regulator of Lyn, acting via CDC2 (Pathan et al, 1996;Wang et al, 1997). Furthermore, the downstream transducer of c-Kit signalling, Stat5a, is negatively regulated by Brca1 (Brizzi et al, 1999;Vidarsson et al, 2002), while Stat1 and Jak2 are positively regulated by both Brca1 and c-Kit Chin et al, 1998;Ouchi et al, 2000;Gao et al, 2001).…”

Induction of apoptosis by Elk-1 and ΔElk-1 proteins

“…The morphology of the DElk-1 transfectants were similar to that of the parental cells. We analysed the expression of Elk-1 and DElk-1 proteins in Elk-1 and DElk-1 transfectants by indirect immuno¯uorescence analysis, immunoperoxidase staining (Figure 1) and Western blot analysis (Figure 2) using Elk-1 polyclonal antibody as described previously Wang et al, 1997). The nuclear and cytoplasmic staining was brighter and stronger in both Elk-1 and DElk-1 transfected cells compared to parental Rat-1 fibroblast cells suggesting higher levels of expression of Elk-1 and DElk-1 proteins in transfected cells ( Figure 1).…”