Nanog Increases Focal Adhesion Kinase (FAK) Promoter Activity and Expression and Directly Binds to FAK Protein to Be Phosphorylated
Nanog Increases Focal Adhesion Kinase (FAK) Promoter Activity and Expression and Directly Binds to FAK Protein to Be Phosphorylated
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DOI：
10.1074/jbc.m111.322883
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发表时间：
2012-05-25
影响因子：
4.8
通讯作者：
Golubovskaya, Vita M.
中科院分区：
文献类型：
--
作者：
Ho, Baotran;Olson, Gretchen;Golubovskaya, Vita M.
研究表明，Nanog和FAK在癌细胞中过表达。在本报告中，Nanog过表达增加了293、SW480和SW620癌细胞中FAK的表达。Nanog结合FAK启动子并上调其活性，而Nanog siRNA则降低FAK启动子活性和FAK mRNA。FAK启动子含有四个Nanog结合位点。这些位点的定点突变显著降低了Nanog对FAK启动子活性的上调。EMSA显示Nanog与这四个位点中的每一个都有特异性结合，芯片分析证实了结合。Nanog通过下拉法和免疫沉淀法直接结合FAK蛋白，并通过共聚焦显微镜进行蛋白质共定位。Nanog结合FAK的N-末端结构域。此外，在体外和体内癌细胞中，FAK以剂量依赖的方式直接磷酸化Nanog。纳米酪氨酸Y35F和Y174F的定点突变阻断了FAK的磷酸化和结合。此外，野生型Nanog的过表达增加了丝足/片状脂肪的形成，而突变体Y35F和Y174F Nanog则没有。野生型Nanog比突变体Y35F和Y174F更能增强细胞侵袭能力，而FAK抑制细胞侵袭的作用更强。用siRNA下调Nanog使细胞生长减少，FAK的过表达逆转了这一趋势。因此，这些数据表明，FAK启动子受Nanog的调控，蛋白的直接结合，FAK对Nanog的磷酸化，以及FAK和Nanog的交叉调节对癌细胞的形态、侵袭和生长的影响，这在癌症发生中起着重要作用。
Nanog and FAK were shown to be overexpressed in cancer cells. In this report, the Nanog overexpression increased FAK expression in 293, SW480, and SW620 cancer cells. Nanog binds the FAK promoter and up-regulates its activity, whereas Nanog siRNA decreases FAK promoter activity and FAK mRNA. The FAK promoter contains four Nanog-binding sites. The site-directed mutagenesis of these sites significantly decreased up-regulation of FAK promoter activity by Nanog. EMSA showed the specific binding of Nanog to each of the four sites, and binding was confirmed by ChIP assay. Nanog directly binds the FAK protein by pulldown and immunoprecipitation assays, and proteins co-localize by confocal microscopy. Nanog binds the N-terminal domain of FAK. In addition, FAK directly phosphorylates Nanog in a dose-dependent manner by in vitro kinase assay and in cancer cells in vivo. The site-directed mutagenesis of Nanog tyrosines, Y35F and Y174F, blocked phosphorylation and binding by FAK. Moreover, overexpression of wild type Nanog increased filopodia/lamellipodia formation, whereas mutant Y35F and Y174F Nanog did not. The wild type Nanog increased cell invasion that was inhibited by the FAK inhibitor and increased by FAK more significantly than with the mutants Y35F and Y174F Nanog. Down-regulation of Nanog with siRNA decreased cell growth reversed by FAK overexpression. Thus, these data demonstrate the regulation of the FAK promoter by Nanog, the direct binding of the proteins, the phosphorylation of Nanog by FAK, and the effect of FAK and Nanog cross-regulation on cancer cell morphology, invasion, and growth that plays a significant role in carcinogenesis.