Deciphering the phosphorylation "code" of the glucocorticoid receptor in vivo.
Deciphering the phosphorylation "code" of the glucocorticoid receptor in vivo.
复制标题
DOI：
10.1074/jbc.m110530200
复制
发表时间：
2002-07-19
影响因子：
4.8
通讯作者：
Garabedian, MJ
中科院分区：
文献类型：
--
作者：
Wang, Z;Frederick, J;Garabedian, MJ
糖皮质激素受体（GR）以激素依赖性方式在多个丝氨酸残基处磷酸化，但由于缺乏检测体内受体磷酸化的简单方法，阐明GR磷酸化功能的进展受到阻碍。我们已经生产了特异性识别人 GR 内 Ser(203) 和 Ser(211) 磷酸化位点的抗体。在没有激素的情况下，与 Ser(203) 处的磷酸化相比，Ser(211) 处的 GR 磷酸化水平较低。 GR 激动剂地塞米松处理后，两个残基的磷酸化均增加。使用一系列激动剂和拮抗剂，我们发现 GR 的转录活性与 Ser(211) 磷酸化量相关，表明 Ser(211) 磷酸化是体内激活 GR 的生物标志物。从机制上讲，Ser(203) 和 Ser(211) 磷酸化响应激素的动力学不同，Ser(211) 相对于 Ser(203) 表现出更稳健和持续的磷酸化。使用磷酸 GR 特异性抗体对 GR 免疫沉淀物进行的分析表明，在没有激素的情况下，受体在 Ser(203) 处被异质化，而在激素存在下，受体亚群在 Ser(203) 和 Ser(211) 处均被磷酸化。有趣的是，激素处理后的生化分级分离研究表明，受体的 Ser(203) 磷酸化形式主要存在于细胞质中，而 Ser(211) 磷酸化 GR 存在于细胞核中。同样，通过免疫荧光，Ser(203)-磷酸化GR位于细胞的细胞质和核周区域，但不在核质中，而在激素处理的细胞的核质中存在明显的强磷酸-Ser(211)染色。我们的结果表明，差异磷酸化的受体种类位于独特的亚细胞区室中，可能调节受体功能的不同方面。
The glucocorticoid receptor (GR) is phosphorylated at multiple serine residues in a hormone-dependent manner, yet progress on elucidating the function of GR phosphorylation has been hindered by the lack of a simple assay to detect receptor phosphorylation in vivo. We have produced antibodies that specifically recognize phosphorylation sites within human GR at Ser(203) and Ser(211). In the absence of hormone, the level of GR phosphorylation at Ser(211) was low compared with phosphorylation at Ser(203). Phosphorylation of both residues increased upon treatment with the GR agonist dexamethasone. Using a battery of agonists and antagonists, we found that the transcriptional activity of GR correlated with the amount of phosphorylation at Ser(211), suggesting that Ser(211) phosphorylation is a biomarker for activated GR in vivo. Mechanistically, the kinetics of Ser(203) and Ser(211) phosphorylation in response to hormone differed, with Ser(211) displaying a more robust and sustained phosphorylation relative to Ser(203). Analysis of GR immunoprecipitates with phospho-GR-specific antibodies indicated that the receptor was phosphorylated heterogeneously at Ser(203) in the absence of hormone, whereas in the presence of hormone, a subpopulation of receptors was phosphorylated at both Ser(203) and Ser(211). Interestingly, biochemical fractionation studies following hormone treatment indicated that the Ser(203)-phosphorylated form of the receptor was predominantly cytoplasmic, whereas Ser(211)-phosphorylated GR was found in the nucleus. Likewise, by immunofluorescence, Ser(203)-phosphorylated GR was located in the cytoplasm and perinuclear regions of the cell, but not in the nucleoplasm, whereas strong phospho-Ser(211) staining was evident in the nucleoplasm of hormone-treated cells. Our results suggest that differentially phosphorylated receptor species are located in unique subcellular compartments, likely modulating distinct aspects of receptor function.