The estrogen receptor a (ERa) is activated as a transcription factor by both estrogen and a large variety of other extracellular signals. The mechanisms of this ligand-independent activation, notably by cAMP signaling, are still largely unknown. We now close the gap in the signaling pathway between cAMP and ERa. Whereas the direct phosphorylation of ERa by the cAMP-activated protein kinase A (PKA) is dispensable, the phosphorylation of the coactivator-associated arginine methyltransferase 1 (CARM1) by PKA at a single serine is necessary and sufficient for direct binding to the unliganded hormone-binding domain (HBD) of ERa, and the interaction is necessary for cAMP activation of ERa. Sustained PKA activity promoting a constitutive interaction may contribute to tamoxifen resistance of breast tumors. Binding and activation involve a novel regulatory groove of the ERa HBD. As a result, depending on the activating signal, ERa recruits different coactivator complexes to regulate alternate sets of target genes.[Keywords: Steroid receptor; signaling; coactivator; protein kinase A; breast cancer; endocrine resistance] Supplemental material is available at http://www.genesdev.org.

The nuclear receptor superfamily comprises ligand-regulated transcription factors that control various developmental and physiological pathways.These receptors share a common modular structure and regulate gene expression through the recruitment of a large set of coregulatory proteins.These transcription cofactors regulate, either positively or negatively, chromatin structure and transcription initiation. One of the first proteins to be identified as a hormone-recruited cofactor was RIP140. Despite its recruitment by agonist-liganded receptors, RIP140 exhibits a strong transcriptional repressive activity which involves several inhibitory domains and different effectors. Interestingly, the RIP140 gene, located on chromosome 21 in humans, is finely regulated at the transcriptional level by various nuclear receptors. In addition, the protein undergoes several post-translational modifications which control its repressive activity. Finally, experiments performed in mice devoid of the RIP140 gene indicate that this transcriptional cofactor is essential for female fertility and energy homeostasis. RIP140 therefore appears to be an important modulator of nuclear receptor activity which could play major roles in physiological processes and hormone-dependent diseases. HistoryIn the early 90's, one of the main goals for several laboratories working on nuclear receptor (NR) signaling was to identify associated proteins which could act as transcriptional coregulators. The efforts were initially focused on partners of the ligand binding domain (LBD) encompassing the ligand-dependent activating function (AF-2) because it was the most convenient (due to the existence of inactivating mutations and to the use of antagonist ligands). RIP140 (Receptor Interacting Protein of 140 kDa) was one of the first NR transcriptional cofactors to be isolated. It was first identified by far-western blotting in human cancer cell extracts using a chimeric radiolabeled probe containing the ligand binding domain (LBD) of the mouse ERα fused to the glutathione-S-transferase [Cavailles et al., 1995]. In the presence of estradiol, this probe detected several bands corresponding to RIP140 and to the p160 family of coactivators. Using the same strategy, the RIP140 cDNA was then isolated from a cDNA expression library established from ZR75-1 breast cancer cells [Cavailles et al., 1995].The mouse RIP140 cDNA was isolated 3 years later from a mouse embryonic library using a yeast two hybrid strategy using the LBD of the orphan TR2 receptor as a bait [Lee et al., 1998]. Currently, the cDNA sequences from several species including rat, dog, chicken, xenopus and zebra fish have been deposited in databases. The RIP140 gene is also known as NRIP1 (Nuclear Receptor-Interacting Protein 1) which is the official symbol provided by the HUGO gene nomenclature committee. Protein domain structureThe human RIP140 protein comprises 1158 amino acids with an overall important identity between species (83% of amino acid identity between human and mouse sequences). When the...

The androgen receptor (AR) is a ligand-activated transcription factor that controls growth and survival of prostate cancer cells. In the present study, we investigated the regulation of AR activity by the receptor-interacting protein 140 (RIP140). We first showed that RIP140 could be coimmunoprecipitated with the receptor when coexpressed in 293T cells. This interaction appeared physiologically relevant because chromatin immunoprecipitation assays revealed that, under R1881 treatment, RIP140 could be recruited to the prostate-specific antigen encoding gene in LNCaP cells. In vitro glutathione S-transferase pull-down assays provided evidence that the carboxy-terminal domain of AR could interact with different regions of RIP140. By means of fluorescent proteins, we demonstrated that ligand-activated AR was not only able to translocate to the nucleus but also to relocate RIP140 from very structured nuclear foci to a diffuse pattern. Overexpression of RIP140 strongly repressed AR-dependent transactivation by preferentially targeting the ligand binding domain-dependent activity. Moreover, disruption of RIP140 expression induced AR overactivation, thus revealing RIP140 as a strong AR repressor. We analyzed its mechanism of transrepression and first demonstrated that different regions of RIP140 could mediate AR-dependent repression. We then showed that the carboxy-terminal end of RIP140 could reverse transcriptional intermediary factor 2-dependent overactivation of AR. The use of mutants of RIP140 allowed us to suggest that C-terminal binding protein played no role in RIP140-dependent inhibition of AR activity, whereas histone deacetylases partly regulated that transrepression. Finally, we provided evidence for a stimulation of RIP140 mRNA expression in LNCaP cells under androgen treatment, further emphasizing the role of RIP140 in androgen signaling.

The orphan receptor short heterodimer partner (SHP) is a common partner for a great number of nuclear receptors, and it plays an important role in many diverse physiological events. In a previous study, we described SHP as a strong repressor of the androgen receptor (AR). Herein, we addressed the mechanism of action of its negative activity on transcription. We first investigated the intrinsic repressive potential of SHP and mapped two core repressive domains to the amino acids 170-210 and 210-240. From GST pull-down assays, we demonstrated a direct interaction between SHP and diverse histone deacetylases (HDACs) as well as a strong interaction between HDAC1 and SHP inhibitory domains. We further supported the evidence for an interaction between SHP and HDAC1 by showing their co-immunoprecipitation and provided evidence for the existence of a ternary complex comprising AR, SHP, and HDAC1. The use of trichostatin A (TSA), a specific inhibitor of HDAC activity, confirmed that HDACs significantly contribute to the intrinsic transrepressive activity of SHP. Finally, we showed that TSA reversed SHP-induced repression of AR, further emphasizing the relevance of the interaction between SHP and HDACs. This latter action affected in a very similar manner SHP-mediated repression of estrogen receptor alpha (ERalpha) transactivation. Altogether, our results indicate that SHP mediates most of its repressive effect through recruitment of HDACs and suggest that the physiological actions of SHP could be affected by HDAC inhibitors.