摘要
Apoptin是一种来源于鸡贫血病毒的小蛋白,在肿瘤细胞中定位于细胞核,而在正常细胞中主要分布于细胞质。根据预测,Apoptin分子中有2段序列(NLS1和NLS2)可能是单组分核定位信号。通过基因突变和缺失构建了Apoptin各种不同的核定位信号突变体和磷酸化突变体,利用增强型绿色荧光蛋白(EGFP)作标签,观察了其在肿瘤细胞中亚细胞定位的变化。结果表明,NLS1和NLS2单独均不是有效的单组分核定位信号。Apoptin的核定位信号是由NLS1和NLS2这2段序列共同组成的双组分核定位信号,缺少任何一段序列都会严重影响Apoptin在肿瘤细胞中的核定位。其中,NLS2对于Apoptin的核定位起主要作用。Apoptin的获得型磷酸化突变体并不能转位到正常细胞的细胞核中,而其磷酸化负突变体仍定位于肿瘤细胞的细胞核。另外,丝氨酸/苏氨酸蛋白激酶抑制剂H7也不影响Apoptin在肿瘤细胞中的核定位。很可能,Apoptin的磷酸化并不参与调控其核定位信号的功能。
Apoptin, a small protein derived from chicken anemia virus, has a nuclear localization in tumor cells, but in normal cells, it has a cytoplastic distribution. It is predicted that Apoptin contains two stretchs of sequence(NLS1 and NLS2) which are putative monopartite nuclear localiztion signals(NLS). Different NLS and phosphorylation mutants tagged by enhanced green fluorescence protein were constructed, and were used to transfect tumor cells to analyze their subcellular distribution. The results showed that both NLS1 and NLS2 are not effective monopartite NLS, respectively. But NLS1 and NLS2 all contribute to effective nuclear translocation of Apoptin in tumor cells, and they are interdependent, yet NLS2 plays a predominant role in the process. In fact, the NLS of Apoptin is a bipartite NLS composed by NLS1 and NLS2. The gain-of-function point mutant of phosphorylation did not tanslocate to the nucleus in normal cells, and the negative mutants of phosphorylation still accumulated in nucleus in tumor cells. On the other hands, H7, a serine/threonine kinase inhibitor, did not inhibit the nuclear translocation of Apoptin in tumor cells. It is possible that phosphorylation dose not regulate the function of NLS of Apoptin.
出处
《生物技术通讯》
CAS
2004年第6期541-545,共5页
Letters in Biotechnology
