The 1,25-dihydroxy-vitamin D3 receptor is phosphorylated in response to 1,25-dihydroxy-vitamin D3 and 22-oxacalcitriol in rat osteoblasts, and by casein kinase II, in vitro.
The 1,25-dihydroxy-vitamin D3 receptor is phosphorylated in response to 1,25-dihydroxy-vitamin D3 and 22-oxacalcitriol in rat osteoblasts, and by casein kinase II, in vitro.
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1,25-二羟基维生素 D3 受体在大鼠成骨细胞中响应 1,25-二羟基维生素 D3 和 22-奥沙骨化三醇，并在体外被酪蛋白激酶 II 磷酸化。
DOI：
10.1021/bi00083a019
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发表时间：
1993
期刊：
影响因子：
2.9
通讯作者：
Haussler,MR
中科院分区：
文献类型：
--
作者：
Jurutka,PW;Terpening,CM;Haussler,MR
摘要：我们分析了大鼠骨肉瘤（ROS 17/2.8）细胞内源性核1,25 -二羟基维生素D3 （1,25 (OH) 2D3）受体（VDR），并提出了生化证据证明它是一种磷酸化蛋白。当ROS 17/2.8细胞用[35S]蛋氨酸代谢标记时，用1 (H M l, 25 (OH) 2D3处理会导致变性聚丙烯酰胺凝胶中免疫沉淀的VDR的电泳迁移率下降，这是磷酸化蛋白的一个特性。用[32P]正磷酸盐对细胞进行类似的标记，会导致32P快速（< 30分钟）、1,25 (OH) 2D3依赖性地结合到54 kda的VDR中，该VDR与从暴露于1,25 (OH) 2D3的[35S]蛋氨酸标记的ROS 17/2.8细胞中提取的迁移速度较慢的受体相结合。对1,25 (OH) 2d3处理过的细胞免疫沉淀的VDR进行碱性磷酸酶处理，可将移动性降低的VDR转化为1,25 (OH) 2d3缺陷细胞中迁移速度更快的VDR。将ROS 17/2.8细胞与非高钙性1,25 (OH) 2D3类似物22-氧骨化三醇（OCT）孵育，产生的VDR磷酸化水平与1,25 (OH) 2D3处理引起的水平相似。用含有从大鼠骨钙素基因中提取的维生素D反应元件的报告载体瞬时转染骨肉瘤细胞，在1,25 (OH) 2D3或oct的存在下产生同等的转录激活，进一步在不同浓度的1,25 (OH) 2D3下进行实验，以评估完整细胞中受体磷酸化和转录活性之间的关系，结果表明这两个参数之间呈正相关。表明1,25 (OH) 2D3激素刺激VDR磷酸化和转录激活并行。最后，高纯度酪蛋白激酶II （CK-II）磷酸化VDR在1,25 (OH) 2d3不依赖的体外反应。比较体内和体外磷酸化的VDR变性凝胶电泳的迁移，发现ck - ii磷酸化受体的电泳迁移率不同，表明ck - ii介导的磷酸化可能不同于响应1,25 (OH) 2D3或oct发生的磷酸化。我们推测，VDR的激素依赖性磷酸化可能是转录激活所必需的，而激素独立的。ck - ii介导的磷酸化可能在受体活性的调节中起作用。
Revised Manuscript Received May 12, 1993 abstract: We analyzed the endogenous nuclear 1, 25-dihydroxy-vitamin D3 (1, 25 (OH) 2D3) receptor (VDR) in rat osteosarcoma (ROS 17/2.8) cells and present biochemical evidence that it is a phosphoprotein. When ROS 17/2.8 cells are labeled metabolically with [35S] methionine, treatment with 1 (H M l, 25 (OH) 2D3 elicits a decrease in the electrophoretic mobility of immunoprecipitated VDR in denaturing polyacrylamide gels, a property characteristic of phosphorylated proteins. Similar labeling of cells with [32P] orthophosphate results in a rapid (< 30 min), l, 25 (OH) 2D3-dependent incorporation of 32P into a 54-kDa VDR species that comigrates with theslower migrating receptor species extracted from [35S] methionine-labeled ROS 17/2.8 cells that have been exposed to l, 25 (OH) 2D3. Alkaline phosphatase treatment of immunoprecipitated VDR from l, 25 (OH) 2D3-treated cells converts the form of the VDR with reduced mobility to the faster migrating species present in l, 25 (OH) 2D3-deficient cells. Incubation of ROS 17/2.8 cells with the non-hypercalcemic l, 25 (OH) 2D3 analog, 22-oxacalcitriol (OCT), produces a level of VDR phosphorylation similar to that elicited by l, 25 (OH) 2D3 treatment. Transient transfection of osteosarcoma cells with a reporter vector containing a vitamin D responsive element derived from the rat osteocalcin gene yields equivalent transcriptional activation in the presence of either l, 25 (OH) 2D3 or OCT. Further experiments performed at various 1, 25 (OH) 2D3 concentrations to assess the relationship between receptor phosphorylation and transcriptional activity in intact cells showed a positive correlation between these two parameters, indicating that the l, 25 (OH) 2D3 hormone stimulates VDR phosphorylation and transcriptional activation in parallel. Finally, highly purified casein kinase II (CK-II) phosphorylates the VDR in a l, 25 (OH) 2D3-independent, in vitro reaction. Comparison of the migration in denaturing gel electrophoresis of in vivo and in vitro phosphorylated VDR reveals a different electrophoretic mobility of the CK-II-phosphorylated receptor, suggesting that CK-II-mediated phosphorylation may be distinct from that occurring in response to l, 25 (OH) 2D3 or OCT. We speculate that hormone-dependent phosphorylation of the VDR may be required for transcriptional activation, while hormone-independent, CK-II-mediated phosphorylation may play a role in the modulation of receptor activity.