Analysis of FK506, timcodar (VX-853) and FKBP51 and FKBP52 chaperones in control of glucocorticoid receptor activity and phosphorylation.
Analysis of FK506, timcodar (VX-853) and FKBP51 and FKBP52 chaperones in control of glucocorticoid receptor activity and phosphorylation.
复制标题
DOI：
10.1002/prp2.76
复制
发表时间：
2014-12
影响因子：
2.6
通讯作者：
Sanchez, Edwin R
中科院分区：
文献类型：
--
作者：
Hinds, Terry D;Stechschulte, Lance A;Elkhairi, Fadel;Sanchez, Edwin R
免疫抑制配体FK506和FK506结合蛋白FKBP52对糖皮质激素受体(GR)活性有刺激作用。在这里，我们通过比较FK506和新的非免疫抑制配体timcodar(VX-853)的GR活性和磷酸化状态，以及在存在和不存在FKBP52和密切相关的蛋白FKBP51的情况下，来探索其潜在的机制。利用FKBP51和FKBP52中的小鼠胚胎成纤维细胞缺陷基因敲除(KO)，我们发现52KO细胞内源基因的GR活性降低，而51KO细胞的GR活性增加。在52KO细胞中，抑制性丝氨酸212处的磷酸化水平升高，而刺激性S220残基的磷酸化水平降低。相反，51KO细胞在刺激残基S220和S234处表现出GR磷酸化增加。在野生型(WT)MEF细胞中，timcodar和FK506一样，在两个内源基因上增强了地塞米松诱导的GR转录活性。在52KO和51KO MEF细胞中，FK506增强了51KO细胞的GR活性，但不能增强52KO细胞的GR活性，提示FKBP52是FK506作用的主要靶点。与FK506一样，替可达增强了51KO细胞的GR，但也增加了52KO细胞的GR活性。FKBP51在52KO细胞中的敲除表明，timcodar的后一种作用需要FKBP51。因此，timcodar似乎对FKBP51和FKBP52具有双重特异性。这项工作证明了磷酸化是FKBP控制GR的一个重要机制，并鉴定了第一个能够靶向GR作用的非免疫抑制大环内酯类化合物。
The immunosuppressive ligand FK506 and the FK506-binding protein FKBP52 are stimulatory to glucocorticoid receptor (GR) activity. Here, we explore the underlying mechanism by comparing GR activity and phosphorylation status in response to FK506 and the novel nonimmunosuppressive ligand timcodar (VX-853) and in the presence and absence of FKBP52 and the closely related protein FKBP51. Using mouse embryonic fibroblast cells (MEFs) deficient knockout (KO) in FKBP51 or FKBP52, we show decreased GR activity at endogenous genes in 52KO cells, but increased activity in 51KO cells. In 52KO cells, elevated phosphorylation occurred at inhibitory serine 212 and decreased phosphorylation at the stimulatory S220 residue. In contrast, 51KO cells showed increased GR phosphorylation at the stimulatory residues S220 and S234. In wild-type (WT) MEF cells, timcodar, like FK506, potentiated dexamethasone-induced GR transcriptional activity at two endogenous genes. Using 52KO and 51KO MEF cells, FK506 potentiated GR activity in 51KO cells but could not do so in 52KO cells, suggesting FKBP52 as the major target of FK506 action. Like FK506, timcodar potentiated GR in 51KO cells, but it also increased GR activity in 52KO cells. Knock-down of FKBP51 in the 52KO cells showed that the latter effect of timcodar required FKBP51. Thus, timcodar appears to have a dual specificity for FKBP51 and FKBP52. This work demonstrates phosphorylation as an important mechanism in FKBP control of GR and identifies the first nonimmunosuppressive macrolide capable of targeting GR action.