Glucocorticoid receptor phosphorylation differentially affects target gene expression
Glucocorticoid receptor phosphorylation differentially affects target gene expression
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DOI：
10.1210/me.2007-0219
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发表时间：
2008-08-01
影响因子：
--
通讯作者：
Garabedian, Michael J.
中科院分区：
文献类型：
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作者：
Chen, Weiwei;Dang, Thoa;Garabedian, Michael J.
糖皮质激素受体（GR）在其N端的多个位点（S203、S211、S226）被磷酸化，但磷酸化在受体功能中的作用尚不清楚。使用一系列激动剂和GR磷酸化位点特异性抗体，我们证明当S211的相对磷酸化超过S226时，GR转录激活最大。与这一发现一致，用丙氨酸替换S226增强GR转录应答。使用一组干扰不同信号传导途径的化合物，我们发现BAPTA-AM（一种细胞内二价阳离子的螯合剂）和姜黄素（一种具有生物活性的天然产物）减少了S211处的激素依赖性磷酸化。GR磷酸化的这种变化与其减少的核保留和转录激活有关。分子模拟表明，GR S211磷酸化促进构象变化，这暴露了一个新的表面可能促进辅因子相互作用。事实上，S211磷酸化增强GR与MED 14（维生素D受体相互作用蛋白150）的相互作用。有趣的是，在表达非磷酸化GR突变体S211 A的U2 OS细胞中，IGF-结合蛋白1和干扰素调节因子8（两者均为MED 14依赖性GR靶基因）的表达相对于表达野生型受体的细胞在广泛的激素浓度范围内降低。相比之下，糖皮质激素诱导的亮氨酸拉链（一种MED 14非依赖性GR靶点）的诱导在高激素水平的S211 A和野生型GR表达细胞中相似，但在低激素浓度的S211 A细胞中减少，表明GR磷酸化、MED 14参与和受体占有之间存在联系。磷酸化也以基因选择性的方式影响GR的抑制程度。因此，GR在S211和S226的磷酸化通过修饰辅因子相互作用来决定GR的转录应答。此外，GR S211磷酸化的作用是基因特异性的，并且在某些情况下，依赖于活化受体的量。
The glucocorticoid receptor (GR) is phosphorylated at multiple sites within its N terminus (S203, S211, S226), yet the role of phosphorylation in receptor function is not understood. Using a range of agonists and GR phosphorylation site- specific antibodies, we demonstrated that GR transcriptional activation is greatest when the relative phosphorylation of S211 exceeds that of S226. Consistent with this finding, a replacement of S226 with an alanine enhances GR transcriptional response. Using a battery of compounds that perturb different signaling pathways, we found that BAPTA- AM, a chelator of intracellular divalent cations, and curcumin, a natural product with antiinflammatory properties, reduced hormone- dependent phosphorylation at S211. This change in GR phosphorylation was associated with its decreased nuclear retention and transcriptional activation. Molecular modeling suggests that GR S211 phosphorylation promotes a conformational change, which exposes a novel surface potentially facilitating cofactor interaction. Indeed, S211 phosphorylation enhances GR interaction with MED14 (vitamin D receptor interacting protein 150). Interestingly, in U2OS cells expressing a nonphosphorylated GR mutant S211A, the expression of IGF- binding protein 1 and interferon regulatory factor 8, both MED14-dependent GR target genes, was reduced relative to cells expressing wildtype receptor across a broad range of hormone concentrations. In contrast, the induction of glucocorticoidinduced leucine zipper, a MED14-independent GR target, was similar in S211A- and wildtype GR- expressing cells at high hormone levels, but was reduced in S211A cells at low hormone concentrations, suggesting a link between GR phosphorylation, MED14 involvement, and receptor occupancy. Phosphorylation also affected the magnitude of repression by GR in a gene- selective manner. Thus, GR phosphorylation at S211 and S226 determines GR transcriptional response by modifying cofactor interaction. Furthermore, the effect of GR S211 phosphorylation is gene specific and, in some cases, dependent upon the amount of activated receptor.