Schweppe R E, Gutierrez-Hartmann A
Department of Biochemistry, Program in Molecular Biology, University of Colorado Health Sciences Center, 4200 East Ninth Avenue, Box B-151, Denver, CO 80262, USA.
Nucleic Acids Res. 2001 Mar 1;29(5):1251-60. doi: 10.1093/nar/29.5.1251.
Ets factors play a critical role in oncogenic Ras- and growth factor-mediated regulation of the proximal rat prolactin (rPRL) promoter in pituitary cells. The rPRL promoter contains two key functional Ets binding sites (EBS): a composite EBS/Pit-1 element located at -212 and an EBS that co-localizes with the basal transcription element (BTE, or A-site) located at -96. Oncogenic Ras exclusively signals to the -212 site, which we have named the Ras response element (RRE); whereas the response of multiple growth factors (FGFs, EGF, IGF, insulin and TRH) maps to both EBSs. Although Ets-1 and GA binding protein (GABP) have been implicated in the Ras and insulin responses, respectively, the precise identity of the pituitary Ets factors that specifically bind to the RRE and BTE sites remains unknown. In order to identify the Ets factor(s) present in GH4 and GH3 nuclear extracts (GH4NE and GH3NE) that bind to the EBSs contained in the RRE and BTE, we used EBS-RRE and BTE oligonucleotides in electrophoretic mobility shift assays (EMSAs), antibody supershift assays, western blot analysis of partially purified fractions and UV-crosslinking studies. EMSAs, using either the BTE or EBS-RRE probes, identified a specific protein-DNA complex, designated complex A, which contains an Ets factor as determined by oligonucleotide competition studies. Using western blot analysis of GH3 nuclear proteins that bind to heparin-Sepharose, we have shown that Ets-1 and GABP, which are MAP kinase substrates, co-purify with complex A, and supershift analysis with specific antisera revealed that complex A contains Ets-1, GABPalpha and GABPbeta1. In addition, we show that recombinant full-length Ets-1 binds equivalently to BTE and EBS-RRE probes, while recombinant GABPalpha/beta preferentially binds to the BTE probe. Furthermore, comparing the DNA binding of GH4NE containing both Ets-1 and GABP and HeLa nuclear extracts devoid of Ets-1 but containing GABP, we were able to show that the EBS-RRE preferentially binds Ets-1, while the BTE binds both GABP and Ets-1. Finally, UV-crosslinking experiments with radiolabeled EBS-RRE and BTE oligonucleotides showed that these probes specifically bind to a protein of approximately 64 kDa, which is consistent with binding to Ets-1 (54 kDa) and/or the DNA binding subunit of GABP, GABPalpha (57 kDa). These studies show that endogenous, pituitary-derived GABP and Ets-1 bind to the BTE, whereas Ets-1 preferentially binds to the EBS-RRE. Taken together, these data provide important insights into the mechanisms by which the combination of distinct Ets members and EBSs transduce differential growth factor responses.
Ets因子在致癌性Ras和生长因子介导的垂体细胞中大鼠催乳素（rPRL）近端启动子的调控中起关键作用。rPRL启动子包含两个关键的功能性Ets结合位点（EBS）：一个位于-212的复合EBS/Pit-1元件和一个与位于-96的基础转录元件（BTE，或A位点）共定位的EBS。致癌性Ras仅向-212位点发出信号，我们将其命名为Ras反应元件（RRE）；而多种生长因子（FGFs、EGF、IGF、胰岛素和TRH）的反应则映射到两个EBS。尽管Ets-1和GA结合蛋白（GABP）分别与Ras和胰岛素反应有关，但特异性结合RRE和BTE位点的垂体Ets因子的确切身份仍然未知。为了鉴定存在于GH4和GH3核提取物（GH4NE和GH3NE）中与RRE和BTE中包含的EBS结合的Ets因子，我们在电泳迁移率变动分析（EMSA）、抗体超迁移分析、部分纯化级分的蛋白质印迹分析和紫外线交联研究中使用了EBS-RRE和BTE寡核苷酸。使用BTE或EBS-RRE探针进行的EMSA鉴定出一种特异性的蛋白质-DNA复合物，命名为复合物A，通过寡核苷酸竞争研究确定其包含一个Ets因子。通过对与肝素-琼脂糖结合的GH3核蛋白进行蛋白质印迹分析，我们表明作为MAP激酶底物的Ets-1和GABP与复合物A共纯化，用特异性抗血清进行的超迁移分析表明复合物A包含Ets-1、GABPα和GABPβ1。此外，我们表明重组全长Ets-1与BTE和EBS-RRE探针的结合能力相当，而重组GABPα/β优先结合BTE探针。此外，比较同时含有Ets-1和GABP的GH4NE与不含Ets-1但含有GABP的HeLa核提取物的DNA结合情况，我们能够表明EBS-RRE优先结合Ets-1，而BTE结合GABP和Ets-两者。最后，用放射性标记的EBS-RRE和BTE寡核苷酸进行的紫外线交联实验表明，这些探针特异性结合一种约64 kDa的蛋白质，这与结合Ets-1（54 kDa）和/或GABP的DNA结合亚基GABPα（57 kDa）一致。这些研究表明，内源性的、垂体来源的GABP和Ets-1与BTE结合，而Ets-1优先与EBS-RRE结合。综上所述，这些数据为不同Ets成员和EBS组合转导不同生长因子反应的机制提供了重要见解。
