Ser84是spindlin1定位与功能发挥的关键位点

Ser84 is The Key Point of Spindlin1 Nuclear Localization and Function

spindlin1为作者所在研究组克隆并命名的肿瘤相关新基因,之前研究表明spindlin1蛋白定位于细胞核,并有可能通过对TCF-4通路的调节参与对肿瘤细胞生长和周期的调控.为进一步探索spindlin1的作用机制,明确spindlin1结构与功能的关系,在生物信息学分析及晶体结构解析的基础上,构建系列突变表达载体,首先以spindlin1蛋白亚细胞定位为指标,观察野生型及系列突变体spindlin1蛋白的亚细胞定位,并进一步检测野生型及突变体spindlin1对TCF-4荧光素酶报告基因转录活性的调控作用,以明确spindlin1定位与功能的关键位点.结果表明:Ser14+Ser84、Ser84+Ser99、Ser14+Ser84+Ser99位点Ser到Ala的联合突变能使野生型融合蛋白在细胞核内集中分布的特性消失,成为全细胞弥散分布,而Ser14、Ser84、Ser99各位点的单独突变或Ser14+Ser99联合突变对spindlin1蛋白的细胞核内分布没有影响.与此同时,对TCF-4荧光素酶报告基因活性的分析表明,Ser14+Ser84、Ser84+Ser99、Ser14+Ser84+Ser99的联合突变使spindlin1对其活性的激活作用消失或降低.上述结果表明,Ser84是spindlin1细胞定位与功能发挥的关键位点,其作用发挥需要Ser14与Ser99的协助.

The previous study suggested that spindlinl is a new tumor related protein which is localized to nuclear and may be involved in regulating cell cycle via TCF-4 pathway. To further study mechanism of spindlinl function, based on the bioinformatics analysis of spindlinl structure, A series of spindlinlwild or mutant expression vectors was constructed to determine the key amino acid points for spindinl localization and function. As previously reported, wild type spindlinl protein was localized in the nuclei of HeLa cells. Cells transfected with construct either with mutation of Ser14＋Ser84, Ser84＋Ser99 or Ser14＋Ser84 ＋Ser99 exhibited a cytoplasm spindlinl expression in a diffused manner, while in those cells transfected with construct with mutation of Serl 4, Ser84, Ser99 or Serl4＋Ser 99, the spinlinl showed a similar subcellular nuclear location as wild type spindlinl. Further TCF-4 reporter assay were performed using these wild type or mutant spindlinl constructs, and the results indicated that mutation of Ser84 with either Serl4 or Ser99 could abolish the promotion of spindlinl on TCF-4 reporter activity. All these result suggested that Ser84 of spindlinl, with the cooperation of Serl4 or Ser99 play a key role in it nuclear localization and its function in regulation of TCF-4 pathway.