Ito Tomomi, Nomura Seiji, Okada Mayumi, Katsumata Yoshinari, Kikkawa Fumitaka, Rogi Tomohiro, Tsujimoto Masafumi, Mizutani Shigehiko
Department of Obstetrics and Gynecology, Nagoya University School of Medicine, Nagoya, 466-8550, Japan.
Biochem Biophys Res Commun. 2002 Jan 25;290(3):1048-53. doi: 10.1006/bbrc.2001.6325.
P-LAP is identical with cystine aminopeptidase as oxytocinase. We previously located on the placental leucine aminopeptidase (P-LAP) gene the footprint site with the high promoter activity (FP3: -214 to -183) and found a possible interaction between it and AP-2 in choriocarcinoma cells. Here, we investigated FP3 in detail and identified the elements responsible for the high basal rate of transcription. Electrophoretic mobility shift assays demonstrated that FP3 would interact with Ikaros as well as AP-2. Further analysis using antibody against Ikaros confirmed that Ikaros was indeed present and bound to FP3. In addition, AP-2alpha and AP-2gamma antibodies supershifted the second complex at FP3. Functionally, mutations that eliminate AP-2 binding reduced promoter activity significantly, while those that eliminate Ikaros binding reduced promoter activity insignificantly. Double mutations of AP-2 and Ikaros decreased promoter activity progressively. We conclude that AP-2 is the main activator and Ikaros functions cooperatively with it for maximal expression of the human P-LAP gene.
胎盘亮氨酸氨肽酶（P-LAP）与作为催产素酶的胱氨酸氨肽酶相同。我们之前在胎盘亮氨酸氨肽酶（P-LAP）基因上定位了具有高启动子活性的足迹位点（FP3：-214至-183），并发现它与绒毛膜癌细胞中的AP-2之间可能存在相互作用。在此，我们详细研究了FP3，并确定了负责高基础转录率的元件。电泳迁移率变动分析表明，FP3会与Ikaros以及AP-2相互作用。使用抗Ikaros抗体的进一步分析证实Ikaros确实存在并与FP3结合。此外，AP-2α和AP-2γ抗体使FP3处的第二个复合物发生超迁移。在功能上，消除AP-2结合的突变显著降低启动子活性，而消除Ikaros结合的突变对启动子活性的降低不显著。AP-2和Ikaros的双重突变逐渐降低启动子活性。我们得出结论，AP-2是主要激活剂，Ikaros与其协同作用以实现人P-LAP基因的最大表达。
