Characterization of the MN/CA 9 promoter proximal region:: a role for specificity protein (SP) and activator protein 1 (AP1) factors
Characterization of the MN/CA 9 promoter proximal region:: a role for specificity protein (SP) and activator protein 1 (AP1) factors
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DOI：
10.1042/0264-6021:3590669
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发表时间：
2001-11-01
影响因子：
4.1
通讯作者：
Kaluz, S
中科院分区：
文献类型：
--
作者：
Kaluzová, M;Pastoreková, S;Kaluz, S
MN/CA IX（MN）是碳酸酐酶家族的肿瘤相关同工酶。先前的MN启动子的缺失分析确定了保护区（PR）1和2对其转录活性至关重要。计算机辅助搜索表明激活蛋白（AP）2和特异性蛋白（SP）1转录因子的假定结合位点，加上PR 1中的CACCC盒和PR 2中的AP 1位点。PR 1与HeLa细胞核提取物在电泳迁移率变动分析（EMSA）中产生四种复合物。其中，三个完全竞争的SP1和转化生长因子-β视网膜母细胞瘤控制元件CACCC盒（RCE）探针，而AP 2探针竞争对相同的三个复合物部分。Supershift EMSA鉴定了复合物1中的SP1和复合物2和4中的SP3。SP1位点的点突变废除了PR 1功能，而影响PR 1中重叠的CACCC盒/AP 2位点的突变对MN启动子活性的影响较小。具有共有结合位点（SP1或AP 2）或RCE代替PR I的块取代的MN启动子突变体证明了PR 1位置的严格选择性，因为只有SP1突变体重建MN启动子活性。共识SP1探针产生相同的SP1和SP3复合物PR 1在EMSA中，因此，我们得出结论，SP活性是必要的和足够的PR 1的位置。通过PR 2复合物与c-Fos抗体的超位移和具有突变的AP 1位点的构建体的活性显著降低，证实了AP 1在PR 2位置的关键作用。详细的缺失分析证明，PR 1 + PR 2占MN启动子活性的90%，而PR 1和PR 2本身都不足以进行反式激活。因此，SP和API因子之间的协同合作分别与相邻的PR 1和PR 2结合，是MN转录活性所必需的。PR 1 + PR 2模块还刺激从异源启动子的转录。用PMA调节API活性可刺激MN表达并激活MN启动子，而抑制蛋白激酶C活性对HeLa细胞MN表达无影响。
MN/CA IX (MN) is a tumour-associated isoenzyme of the carbonic anhydrase family. Previous deletion analysis of the MN promoter established that protected regions (PRs) 1 and 2 are crucial for its transcriptional activity. Computer-assisted searching indicated putative binding sites for activator protein (AP) 2 and specificity protein (SP) 1 transcription factors, plus a CACCC box in PR1 and an AP1 site in PR2. PR1 produced four complexes in electrophoretic mobility-shift assay (EMSA) with HeLa nuclear extracts. Of these, three were completely competed with the SP1 and transforming growth factor-beta retinoblastoma control-element CACCC box (RCE) probes, whereas the AP2 probe competed against the same three complexes partially. Supershift EMSA identified SP I in the complex 1 and SP3 in the complexes 2 and 4. Point mutations in the SP1 site abrogated the PR1 function, while mutations affecting the overlapping CACCC box/AP2 site in PR1 had minor effect on MN promoter activity. Block-replaced MN promoter mutants that had a consensus binding site (SP1 or AP2) or the RCE in place of PR I demonstrated the stringent selectivity of the PR1 position as only the SP1 mutant reconstituted the MN promoter activity. The consensus SP1 probe generated the same SP1 and SP3 complexes as PR1 in EMSA; therefore we conclude that SP activity is both necessary and sufficient in the PR1 position. The critical role of AP1 in the PR2 position was confirmed by supershift of the PR2 complex with c-Fos antibody and markedly decreased activity of the construct with a mutated AP1 site. Detailed deletion analysis proved that PR1 + PR2 account for 90% of the MN promoter activity, while neither PR I nor PR2 on their own are sufficient for trans activation. Thus, synergistic co-operation between SP and API factors bound to the adjacent PR1 and PR2, respectively, is necessary for MN transcriptional activity. The PR1 + PR2 module also stimulated transcription from a heterologous promoter. The modulation of API activity with PMA stimulated MN expression and activated the MN promoter, whereas inhibition of protein kinase C activity had no effect on MN expression in HeLa cells.