Lazennec G, Thomas J A, Katzenellenbogen B S
Department of Molecular and Integrative Physiology, University of Illinois at Urbana-Champaign, 524 Burrill Hall, 407 South Goodwin Ave, Urbana, IL 61801, USA.
J Steroid Biochem Mol Biol. 2001 Jun;77(4-5):193-203. doi: 10.1016/s0960-0760(01)00060-7.
Estrogen receptor (ER) and cAMP signaling pathways interact in a number of estrogen target tissues including mammary and uterine tissues. One aspect of this interaction is that estradiol and protein kinase A (PKA) activators can cooperate synergistically to activate ER-mediated transcription of both endogenous genes and reporter genes containing only estrogen response elements. The purpose of this study was to investigate the molecular mechanism of this interaction between signaling pathways. Site-directed mutagenesis of the potential PKA phosphorylation sites in the ER indicated that phosphorylation of these sites was not necessary for the observed transcriptional synergy. In transient transfection assays in two different cell lines using reporter constructs containing either cAMP response elements, estrogen response elements or both types of elements, with the addition or absence of cAMP response element binding protein (CREB) expression plasmid, we observed that only one of these cell lines exhibited estrogen/PKA transcriptional synergy. Experiments demonstrated that CREB itself was involved in the transcriptional synergy, and that transfection of CREB restored transcriptional synergy in the cell line in which it was lacking. A functional interaction between ER and CREB was also demonstrated using a mammalian cell protein interaction assay; a dominant negative mutant of CREB did not exhibit this interaction. Therefore, these data indicate that CREB protein is required for the transcriptional synergy between cAMP and estrogen signaling pathways. Furthermore, CREB cooperated with the ER on genes that did not contain cAMP response elements, but contained only estrogen response elements. We propose that activated CREB is recruited to estrogen responsive genes by an ER--coactivator complex containing proteins such as CREB binding protein (CBP) and that the interaction of CREB with ER may assist in stabilizing its interaction with CBP and in promoting estrogen-ER and PKA transcriptional synergy.
雌激素受体（ER）和环磷酸腺苷（cAMP）信号通路在包括乳腺和子宫组织在内的多种雌激素靶组织中相互作用。这种相互作用的一个方面是，雌二醇和蛋白激酶A（PKA）激活剂可以协同激活内源性基因和仅含有雌激素反应元件的报告基因的ER介导转录。本研究的目的是探讨信号通路之间这种相互作用的分子机制。对ER中潜在的PKA磷酸化位点进行定点诱变表明，这些位点的磷酸化对于观察到的转录协同作用并非必需。在两种不同细胞系中进行的瞬时转染实验中，使用含有cAMP反应元件、雌激素反应元件或两种元件的报告构建体，并添加或不添加cAMP反应元件结合蛋白（CREB）表达质粒，我们观察到只有其中一种细胞系表现出雌激素/PKA转录协同作用。实验表明，CREB本身参与了转录协同作用，并且转染CREB可恢复缺乏该蛋白的细胞系中的转录协同作用。使用哺乳动物细胞蛋白相互作用试验也证明了ER和CREB之间的功能相互作用；CREB的显性负突变体未表现出这种相互作用。因此，这些数据表明，CREB蛋白是cAMP和雌激素信号通路之间转录协同作用所必需的。此外，CREB在不含cAMP反应元件但仅含有雌激素反应元件的基因上与ER协同作用。我们提出，活化的CREB通过含有CREB结合蛋白（CBP）等蛋白质的ER-共激活因子复合物被招募到雌激素反应基因上，并且CREB与ER的相互作用可能有助于稳定其与CBP的相互作用，并促进雌激素-ER和PKA转录协同作用。
