Cloning and characterization of the promoter region of human focal adhesion kinase gene: nuclear factor kappa B and p53 binding sites
Cloning and characterization of the promoter region of human focal adhesion kinase gene: nuclear factor kappa B and p53 binding sites
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DOI：
10.1016/j.bbaexp.2004.03.002
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发表时间：
2004-05-25
期刊：
影响因子：
--
通讯作者：
Cance, W
中科院分区：
文献类型：
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作者：
Golubovskaya, V;Kaur, A;Cance, W
粘着斑激酶（FAK）基因编码的粘着斑激酶定位于细胞与细胞外基质的接触点。研究表明，FAK表达在多种恶性肿瘤中增加，在肿瘤发生的早期和晚期阶段。为了了解FAK基因表达和调控的机制，我们克隆并鉴定了FAK基因的5'启动子区。将带有FAK启动子的1.2kb片段置于pGL 3-Basic载体中的荧光素酶报告基因的上游，并转染到不同的细胞系中。内源性高FAK表达细胞系与低FAK表达细胞相比显示高水平的荧光素酶活性，表明FAK在转录水平上调节。序列缺失构建体显示，最大FAK启动子活性需要类似于600个碱基对的区域（类似于564至+47）。FAK的5 '侧翼区富含GC，含有几个潜在的转录因子结合位点，包括两个NF-κ B和p53结合位点。用NF-κ B超阻遏物抑制NF-κ B可降低FAK荧光素酶活性。用TNF-α诱导增加荧光素酶活性，证实了NF-κ B转录因子在FAK转录激活中的作用。电泳迁移率变动分析（EMSA）显示NF-κ B和p53转录因子与FAK启动子区结合。NF-κ B和p53质粒与FAK启动子荧光素酶构建体的共转染分别证明了FAK荧光素酶活性的诱导和抑制。这一结果为进一步研究FAK的转录调控机制提供了分子基础。(C)2004 Elsevier B. V.保留所有权利。
Focal adhesion kinase (FAK) gene encodes focal adhesion kinase that localizes at contact points of cells with extracellular matrix. It was shown that FAK expression is increased in a variety of malignancies, both at early and advanced stages of tumorigenesis. To understand mechanisms of FAK gene expression and regulation, we cloned and characterized the 5' promoter region of the FAK gene. The 1.2-kb fragment with FAK promoter was placed upstream of the luciferase reporter gene in a pGL3-Basic vector and transfected into different cell lines. Endogenous high-FAK-expressing cell lines showed high levels of luciferase activity in contrast to low-FAK-expressing cells, indicating on transcriptional level of FAK regulation. Serial deletion constructs revealed that a similar to 600 base pair region (similar to 564 to + 47) is required for the maximal FAK promoter activity. The 5'-flanking region of FAK is GC-rich and contains several potential transcription factor binding sites, including two NF-kappa B and p53 binding sites. Inhibition of NF-kappa B with NF-kappa B super-repressor decreased FAK luciferase activity. Induction with TNF-alpha increased luciferase activity confirming a role of NF-kappa B transcription factor in the FAK transcriptional activation. The binding of NF-kappa B and p53 transcription factors to the FAK promoter region was demonstrated by electrophoretic mobility shift assay (EMSA). Cotransfection of NF-kappa B and p53 plasmids with FAK promoter luciferase constructs demonstrate induction and inhibition, respectively, of FAK luciferase activity. The results provide a molecular basis for analysis of FAK transcriptional regulation. (C) 2004 Elsevier B.V. All rights reserved.