CONSEQUENCES OF ACETYLATION OF ESTROGEN RECEPTOR ALPHA BY P300

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目及 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 使用各种生物化学和细胞为基础的方法，我们表明，雌激素受体α（ER α）是乙酰化的p300乙酰化酶的配体和类固醇受体辅激活剂依赖性的方式。使用诱变和质谱，我们确定了两个保守的赖氨酸残基ER α（Lys 266和Lys 268）的p300介导的乙酰化的主要目标。这些残基在细胞中被乙酰化，如使用特异性识别在Lys 266和Lys 268处乙酰化的ER α的抗体通过免疫沉淀-Western印迹实验所确定的。通过p300的ER α的乙酰化被天然细胞脱乙酰酶逆转，包括抑制素A敏感性酶（即I类和II类脱乙酰酶）和烟酰胺腺嘌呤二核苷酸依赖性/烟酰胺敏感性酶（即III类脱乙酰酶，如沉默调节蛋白1）。Lys 266和Lys 268处的乙酰化，或用谷氨酰胺（即K266/268 Q）（一种模拟乙酰化赖氨酸的残基）取代相同残基，可增强EMSA中ER α的DNA结合活性。同样，在基于细胞的报告基因测定中，用谷氨酰胺取代Lys 266和Lys 268增强ER α的配体依赖性活性。总的来说，我们的研究结果暗示乙酰化作为ER α的配体依赖性基因调控活性的调节剂。这种调节可能在正常和病理状态下的各种雌激素靶组织中的雌激素依赖性信号传导结果中发挥作用。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Using a variety of biochemical and cell-based approaches, we show that estrogen receptor alpha (ERalpha) is acetylated by the p300 acetylase in a ligand- and steroid receptor coactivator-dependent manner. Using mutagenesis and mass spectrometry, we identified two conserved lysine residues in ERalpha (Lys266 and Lys268) that are the primary targets of p300-mediated acetylation. These residues are acetylated in cells, as determined by immunoprecipitation-Western blotting experiments using an antibody that specifically recognizes ERalpha acetylated at Lys266 and Lys268. The acetylation of ERalpha by p300 is reversed by native cellular deacetylases, including trichostatin A-sensitive enzymes (i.e. class I and II deacetylases) and nicotinamide adenine dinucleotide-dependent/nicotinamide-sensitive enzymes (i.e. class III deacetylases, such as sirtuin 1). Acetylation at Lys266 and Lys268, or substitution of the same residues with glutamine (i.e. K266/268Q), a residue that mimics acetylated lysine, enhances the DNA binding activity of ERalpha in EMSAs. Likewise, substitution of Lys266 and Lys268 with glutamine enhances the ligand-dependent activity of ERalpha in a cell-based reporter gene assay. Collectively, our results implicate acetylation as a modulator of the ligand-dependent gene regulatory activity of ERalpha. Such regulation is likely to play a role in estrogen-dependent signaling outcomes in a variety of estrogen target tissues in both normal and pathological states.