Blind Raymond D, Garabedian Michael J
Department of Microbiology, NYU Cancer Institute, NYU School of Medicine, 550 First Avenue, New York, NY 10016, United States.
J Steroid Biochem Mol Biol. 2008 Mar;109(1-2):150-7. doi: 10.1016/j.jsbmb.2008.01.002. Epub 2008 Jan 19.
The human glucocorticoid receptor (GR) is phosphorylated on its N-terminus at three major sites (S203, S211 and S226) within activation function 1 (AF1). Although GR has been shown to assemble at glucocorticoid responsive elements (GREs) in the presence of hormone, the timing and specificity of GR phospho-isoform recruitment to receptor target genes has not been established. Using chromatin immunoprecipitation (ChIP) and GR phosphorylation site-specific antibodies, we examined GR phospho-isoform recruitment to several glucocorticoid-induced genes including tyrosine aminotransferase (tat) and sulfonyltransferase-1A1 (sult) in rat hepatoma cells, and the glucocorticoid-induced leucine zipper (gilz) gene in human U2OS cells. GR P-S211 and GR P-S226 isoforms were efficiently recruited to the tat, sult and gilz GREs in a hormone-dependent manner. In contrast, the GR P-S203 isoform displayed no significant recruitment to any GREs of the genes analyzed, consistent with its lack of nuclear accumulation. Interestingly, the kinetics of GR P-S211 and GR P-S226 recruitment differed among genes. Our findings indicate that GR phospho-isoforms selectively occupy GR target genes, and suggests gene specific requirements for GR phosphorylation in receptor-dependent transcriptional activation.
人类糖皮质激素受体（GR）在其激活功能1（AF1）的N端三个主要位点（S203、S211和S226）发生磷酸化。尽管已证明GR在激素存在的情况下会在糖皮质激素反应元件（GREs）处组装，但GR磷酸化异构体募集到受体靶基因的时间和特异性尚未确定。我们使用染色质免疫沉淀（ChIP）和GR磷酸化位点特异性抗体，检测了GR磷酸化异构体募集到大鼠肝癌细胞中几个糖皮质激素诱导基因，包括酪氨酸转氨酶（tat）和磺基转移酶-1A1（sult），以及人U2OS细胞中糖皮质激素诱导的亮氨酸拉链（gilz）基因的情况。GR P-S211和GR P-S226异构体以激素依赖的方式有效地募集到tat、sult和gilz的GREs处。相比之下，GR P-S203异构体对所分析基因的任何GREs均未显示出明显的募集，这与其缺乏核积累一致。有趣的是，GR P-S211和GR P-S226募集的动力学在不同基因之间有所不同。我们的研究结果表明，GR磷酸化异构体选择性地占据GR靶基因，并提示在受体依赖性转录激活中，基因对GR磷酸化有特定要求。
