Ikonen T, Palvimo J J, Kallio P J, Reinikainen P, Jänne O A
Department of Physiology, University of Helsinki, Finland.
Endocrinology. 1994 Oct;135(4):1359-66. doi: 10.1210/endo.135.4.7925097.
The effect of modulators of protein phosphorylation on the transcriptional activity of the androgen receptor (AR) was studied under transient expression conditions. Activators of protein kinase-A [8-bromo-cAMP (8-Br-cAMP)] and protein kinase-C (phorbol 12-myristate 13-acetate) or an inhibitor of protein phosphatase-1 and -2A (okadaic acid) influenced minimally pMMTV-chloramphenicol acetyl-transferase (CAT) activity in CV-1 cells cotransfected with an AR expression plasmid in the absence of androgen. In the presence of testosterone, however, all compounds enhanced AR-mediated transactivation by 2- to 4-fold. A nonsteroidal antiandrogen, Casodex, behaved as a pure antagonist; it blunted the action of testosterone and was not rendered agonistic by activators of protein kinase-A. A reporter plasmid containing two androgen response elements (AREs) in front of the thymidine kinase promoter (pARE2tk-CAT) was also used to examine promoter specificity. It was activated by 8-Br-cAMP, forskolin, or okadaic acid even without AR or androgen. However, when forskolin or okadaic acid was used together with androgen and AR, the resulting AR-dependent transactivation of pARE2tk-CAT was more than additive. Intact DNA- and ligand-binding domains, but not the N-terminal amino acid residues 40-147, of the receptor were mandatory for the synergism between protein kinase-A activators and androgen. Immunoreactive AR content in transfected COS-1 cells was not influenced by exposure to 8-Br-cAMP. Similar results were obtained by ligand binding assays. Quantitative or qualitative differences were not observed in DNA-binding characteristics between receptors extracted from cells treated with testosterone with or without protein kinase-A activator. Collectively, the synergistic stimulation of AR-dependent transactivation by androgen and protein kinase activators is not due to changes in cellular AR content or affinity of the receptor for the cognate DNA element; rather, this phenomenon seems to result from altered interaction of ligand-activated AR with other proteins in the transcription machinery.
在瞬时表达条件下，研究了蛋白质磷酸化调节剂对雄激素受体（AR）转录活性的影响。蛋白激酶-A的激活剂[8-溴环磷酸腺苷（8-Br-cAMP）]和蛋白激酶-C（佛波醇12-肉豆蔻酸酯13-乙酸酯）或蛋白磷酸酶-1和-2A的抑制剂（冈田酸）在无雄激素的情况下，对与AR表达质粒共转染的CV-1细胞中pMMTV-氯霉素乙酰转移酶（CAT）活性的影响极小。然而，在睾酮存在的情况下，所有化合物均使AR介导的反式激活增强了2至4倍。一种非甾体类抗雄激素药物，比卡鲁胺，表现为纯粹的拮抗剂；它削弱了睾酮的作用，并且不会因蛋白激酶-A的激活剂而产生激动作用。还使用了在胸苷激酶启动子（pARE2tk-CAT）前含有两个雄激素反应元件（AREs）的报告质粒来检测启动子特异性。即使没有AR或雄激素，它也能被8-Br-cAMP、福斯高林或冈田酸激活。然而，当福斯高林或冈田酸与雄激素和AR一起使用时，所产生的pARE2tk-CAT的AR依赖性反式激活作用大于相加效应。受体完整的DNA和配体结合结构域，但不是N端40-147氨基酸残基，对于蛋白激酶-A激活剂与雄激素之间的协同作用是必需的。转染的COS-1细胞中的免疫反应性AR含量不受暴露于8-Br-cAMP的影响。通过配体结合试验也获得了类似的结果。在用或不用蛋白激酶-A激活剂处理睾酮的细胞中提取的受体之间，在DNA结合特性方面未观察到定量或定性差异。总体而言，雄激素和蛋白激酶激活剂对AR依赖性反式激活的协同刺激不是由于细胞AR含量的变化或受体对同源DNA元件的亲和力的改变；相反，这种现象似乎是由于配体激活的AR与转录机制中的其他蛋白质之间相互作用的改变所致。
