In this case, phosphorylation of SRC-3 has a biphasic effect on retinoic acid receptor- transactivation with facilitation followed by restriction of transcription

In this case, phosphorylation of SRC-3 has a biphasic effect on retinoic acid receptor- transactivation with facilitation followed by restriction of transcription. Because the presence of PR is required for SRC-1 degradation, two important remaining questions concern the identification of the key (S)-GNE-140 player responsible for SRC-1 degradation and whether this factor is involved in both basal and ligand-induced SRC-1 down-regulation. demonstrated by confocal imaging. Moreover, SRC-1 was stabilized in the presence of leptomycin B or several proteasomal inhibitors. Two degradation motifs, amino-acids 2C16 corresponding to a PEST motif and amino acids 41C136 located in the basic helix loop helix domain of the coactivator, were identified and shown to control the stability as well as the hormone-dependent down-regulation of the coactivator. SRC-1 degradation is of physiological importance because the two nondegradable mutants that still interacted with PR as demonstrated by coimmunoprecipitation failed to stimulate transcription of exogenous and endogenous target genes, suggesting that concomitant PR/SRC-1 ligand-dependent degradation is (S)-GNE-140 a necessary step for PR transactivation activity. Collectively our findings are consistent with the emerging role of proteasome-mediated proteolysis in the gene-regulating process and indicate that the ligand-dependent down-regulation of SRC-1 is critical for PR transcriptional activity. The progesterone receptor (PR), also known as NR3C3, plays a crucial role in the coordination of several aspects of female reproductive development and function (1). Invalidation of the gene in mice leads to pleiotropic reproductive abnormalities and demonstrates that PR orchestrates key events associated with the establishment and maintenance of pregnancy. From a pathophysiological perspective, accumulating evidence indicates that PR is involved in breast cancer Rabbit Polyclonal to STAT3 (phospho-Tyr705) cells proliferation and is implicated in the development and progression of breast cancer (2). Coregulators (coactivators or corepressors) are important nuclear receptor (NR)-recruited cofactors modulating NR-mediated transcription and leading to activation or repression of target specific genes (3). Steroid receptor coactivator-1 (SRC-1) is a PR coactivator belonging to the p160 gene family, which contains three homologous members (SRC-1, -2, and -3) serving as NR transcriptional coactivators (4). This family of coactivators is characterized by the presence of several conserved functional domains: a basic helix-loop-helix (bHLH)-Per-ARNT-Sim N-terminal domain, a cAMP response element-binding protein (CBP) interacting domain (AD1), a glutamine-rich region, a C-terminal activation domain (AD2), and several Lrepresent the intensity (S)-GNE-140 profile for the proteasome antigen S7/Rpt1 signal, and the represent the (S)-GNE-140 intensity profile for SRC-1 signal. refer to identified speckles: cytoplasmic (1 to 7) or nuclear (8 to 11). Note that although the fluorescence intensity from the two channels is different, the peaks of both signals are overlapping. SRC-1 is ubiquitinylated and is degraded by the proteasome We next studied the mechanism of SRC-1 down-regulation. First, we investigated whether the coactivator was ubiquitinylated and targeted to the proteasome. COS-7 cells were transfected with the expression vector encoding the full-length SRC-1 and incubated in the presence of proteasome inhibitors, MG132, or epoxomicin. Consistent with previous reports (14, 35), both inhibitors increased SRC-1 protein level in comparison with cells treated with vehicle (Fig. 2A and Supplemental Fig. 3). To demonstrate that SRC-1 is polyubiquitinylated, COS-7 cells were transfected with SRC-1 expression vector in the presence or absence of a vector encoding His-tagged ubiquitin (His 6-Ub) and analyzed by Western blot. In the absence of His 6-Ub, the anti-SRC-1 antibody detected a major band of approximately 160 kDa (Fig. 2B, (41) have shown that upon ligand treatment, PR preferentially interacts with SRC-1. (S)-GNE-140 We thus investigated whether SRC-1 down-regulation might be also modulated by PR ligands. As previously reported (22), immunocytochemical studies (Fig. 3A) and Western blot experiments (Supplemental Fig. 5) showed that the agonist ligand R5020 stimulates stably expressed endogenous PR proteolysis after 24 h treatment, whereas the antagonist ligand RU486 prevents PR proteolysis in Ishikawa cells stably expressing PR-B (Ishi-PR-B). To test the impact of ligands on SRC-1 expression level, Ishi-PR-B cells were transiently transfected with a SRC-1 expression vector and incubated overnight with R5020 or RU486. Western blot analyses revealed that SRC-1 and PR are concomitantly degraded in the presence of agonist R5020 and that RU486 prevents the degradation.