BRCA1 Regulates Acetylation and Ubiquitination of Estrogen Receptor-α

Abstract

Inherited mutations of the breast cancer susceptibility gene BRCA1 confer a high risk for breast cancer development. The (300)RXKK and (266)KXK motifs have been identified previously as sites for acetylation of the estrogen receptor-alpha (ER-alpha), and (302)K was also found to be a site for BRCA1-mediated mono-ubiquitination of ER-alpha in vitro. Here we show that ER-alpha proteins with single or double lysine mutations of these motifs (including K303R, a cancer-associated mutant) are resistant to inhibition by BRCA1, even though the mutant ER-alpha proteins retain the ability to bind to BRCA1. We also found that BRCA1 overexpression reduced and knockdown increased the level of acetylated wild-type ER-alpha, without changing the total ER-alpha protein level. Increased acetylation of ER-alpha due to BRCA1 small interfering RNA was dependent upon phosphatidylinositol 3-kinase/Akt signaling and on up-regulation of the coactivator p300. In addition, using an in vitro acetylation assay, we found that in vitro-translated wild-type BRCA1 but not a cancer-associated point mutant (C61G) inhibited p300-mediated acetylation of ER-alpha. Furthermore, BRCA1 overexpression increased the levels of mono-ubiquitinated ER-alpha protein, and a BRCA1 mutant that is defective for ubiquitin ligase activity but retains other BRCA1 functions (I26A) did not ubiquitinate ER-alpha or repress its activity in vivo. Finally, ER-alpha proteins with mutations of the (300)RXKK or (266)KXK motifs showed modest or no BRCA1-induced ubiquitination. We propose a model in which BRCA1 represses ER-alpha activity, in part, by regulating the relative degree of acetylation vs. ubiquitination of ER-alpha.

Phosphorylation by p38 Mitogen-Activated Protein Kinase Promotes Estrogen ReceptorαTurnover and Functional Activity via the SCFSkp2Proteasomal Complex

“…The purification of the GST-fused ER␣ protein from cultures of bacteria grown overnight and GST pulldown assays thereafter were performed as described previously (20). The in vitro translation of Skp2A and Skp2B and the incorporation of 35 S-labeled methionine (Perkin-Elmer) were performed by use of the TNT T7-coupled reticulocyte lysate system kit (Promega). Labeled Skp2A or Skp2B and GST-ER␣ (or GST alone) were incubated with glutathione-Sepharose beads (Amersham) in binding buffer (20 mM Tris [pH 7.5], 50 nM NaCl, 10% glycerol, 10 mM NaF, 1% Nonidet P-40, 1 mM NaVO 4 , 1ϫ protease inhibitor cocktail [Roche], and 1 mM phenylmethylsulfonyl fluoride [PMSF]) at 4°C for 4 h with gentle rotation.…”

RETRACTED ARTICLE: Regulation of estrogen receptor signaling in breast carcinogenesis and breast cancer therapy

“…Both BRCA1 and BRCA2 are E2-responsive genes and BRCA1 in turn regulates ERα activity through posttranslational mechanisms [125,126]. Loss of BRCA1 expression also causes cells to acquire tamoxifen resistance [127] due to the increased levels of coactivator and the decreased recruitment of corepressors onto ERα-regulated gene promoters under BRCA1 silencing.…”

“…2). [152], E6-associated protein (e6AP) [153], RING finger protein 147 (RNF147, also named estrogen-responsive finger protein (EFP)) [154], SPOP [155], CHIP [156][157][158] and BRCA1/BARD1 [126,159] regulate ERα proteasomal degradation (Fig. 2).…”

“…The BRCA1/BARD1 complex has been observed to monoubiquitinate and polyubiquitinate proteins. ERα has also been shown to be a substrate for BRCA1/BARD1 in vitro and in vivo [126,159,163]. BRCA1/BARD1 monoubiquitinates ERα at Lys302, which is a residue located at the C-terminal ligand binding domain.…”

“…E3 ligase activity of BRCA1/BARD1 appears to inhibit estrogen-stimulated ERα transcriptional activity. Overexpression of BRCA1 strongly inhibits ERα activity in MCF-7 cells; whereas the BRCA1-I26A mutant, which inhibits BRCA1/BARD1 ubiquitin ligase activity, is resistant to repressive effect on ERα activity without affecting interaction between BRCA1 and ERα [126]. It remains to be determined whether BRCA1 monoubiquitination leads to further polyubiquitination and the consequential proteasomal degradation of ERα.…”