Structural determinants of the BRCA1 : estrogen receptor interaction

Abstract

Previously, we showed that the BRCA1 protein interacts directly and functionally with estrogen receptor-alpha (ER-a), resulting in the inhibition of estradiol (E2)-stimulated ER-a transcriptional activity. The interaction sites were mapped to the N-terminus of BRCA1 (within amino acids (aa) 1-302) and the ligand-binding domain/ activation function-2 (LBD/AF-2) region (within aa 282-420) of ER-a. In this study, we have further characterized the structure/function relationship for the BRCA1 : ER-a interaction. We found that the N-terminal RING domain (aa 20-64) is not required for the BRCA1 : ER-a interaction. We identified two separate contact points for ER-a, one within aa 1-100 and the other within aa 100-200 of BRCA1; and we showed that each of these BRCA1 peptides interacts with BRCA1 in vitro and in vivo. By using different fragments of the BRCA1 Nterminus, we found that aa 67-100 and 101-133 are required for the interaction with ER-a, but that aa 1-67 and 134-302 are dispensible. Previously, we showed that BRCA1 aa 1-302 does not inhibit E2-stimulated ER-a transcriptional activity but does bind to ER-a and acts as a dominant negative inhibitor of the full-length BRCA1 protein. Somewhat surprisingly, we found that BRCA1 aa 1-100 and BRCA1 aa 101-200 (but not aa 201-300) each inhibited ER-a activity, although not as efficiently as fulllength BRCA1. Mutations within an HIV Rev-like nuclear export signal that resembles a nuclear receptor corepressor motif (aa 86-95) impaired the ability of both truncated (aa 1-100) and full-length (aa 1-1863) BRCA1 proteins to interact with and/or repress ER-a activity. Based on these findings, a partial BRCA1 : ER-a threedimensional structure is proposed. The implications of these findings for understanding the BRCA1 : ER-a interaction are discussed.

Estrogen receptor α is a putative substrate for the BRCA1 ubiquitin ligase

“…2 A), suggesting mutation of I358 and Q375 in the ER␣ coactivator-binding region decreases interactions with BRCA1/BARD1 and therefore decreases ubiquitination. An interaction between BRCA1/BARD1 and the ER␣ coactivator-binding region is consistent with previous studies showing peptides containing the ER␣-LBD coactivator-binding region can interact with BRCA1 302 (28). ER␣-I358A/Q375A is native-like, as determined by near-and far-UV CD and a thermal melt similar to WT (data not shown).…”

“…This inhibition has been suggested to occur from an interaction between BRCA1 302 and ER␣-LBD (16,28). These studies found that the BRCA1 RING domain (residues 1-67) is not required for the observed interaction with ER␣, and that the cancer-predisposing mutation, C61G, in full-length BRCA1, does not disrupt the BRCA1-ER␣ interaction.…”

BRCA1 in hormonal carcinogenesis: basic and clinical research

“…The ER-a side of the interacting surface is an a-helix of ER-a (amino acids 338-379), which is at the opposite side of the ER-a homodimerization interface. Interestingly, two tumorassociated BRCA1 mutations at the interacting surface (L63F and I89T) were found to impair the ability of BRCA1 to repress ER-a activity (Ma et al 2005). Zheng and colleagues (2001) demonstrated constitutive activation of exogenous ER-a in the Brca1-null mouse embryo fibroblasts (MEFs), suggesting that the endogenous BRCA1 protein may mediate the ligandindependent repression of ER-a.…”

“…A more detailed study of the BRCA1:ER-a interaction revealed two potential contact sites for BRCA1 on ER-a (the major site within amino acids 338-379 and a minor site within amino acids 420-595) and two contact sites for ER-a on BRCA1 (amino acids 67-100 and 101-134) (Ma et al 2005). BRCA1 contains a conserved helical motif (amino acids 86-95) resembling a previously identified nuclear corepressor motif (Lxx(I/ H)Ixxx(I/L), where x = any amino acid), mutation of which disrupted the ability of BRCA1 to bind and repress ER-a (Ma et al 2005).…”

“…BRCA1 contains a conserved helical motif (amino acids 86-95) resembling a previously identified nuclear corepressor motif (Lxx(I/ H)Ixxx(I/L), where x = any amino acid), mutation of which disrupted the ability of BRCA1 to bind and repress ER-a (Ma et al 2005). Based on these studies, a partial BRCA1: ER-a three-dimensional structure was proposed (Fig.…”