双亮氨酸模体对NOK/STYK1蛋白的表达及细胞内分布特征的影响

Di-leucine Motifs of NOK/STYK1 Affects Its Protein Expression and Distribution in HeLa Cells

NOK(novel oncogene with kinase domain)蛋白是一种新的在结构上比较独特的受体型酪氨酸蛋白激酶家族(protein tyrosine kinases,PTKs)成员,具有较强的促肿瘤形成及转移的能力,并参与细胞的内吞过程。基因结构序列分析结果表明,在NOK氨基酸序列中存在多处双亮氨酸模体(di-leucine motif)。有研究表明,双亮氨酸模体在细胞的内吞、膜泡运输以及细胞信号分拣等过程中具有重要作用。为此,该研究构建了一系列NOK蛋白双亮氨酸模体突变体,并在He La细胞中分析了其蛋白质表达水平及细胞内分布特征。结果表明,NOK蛋白的不同双亮氨酸模体的突变可导致NOK蛋白在细胞中表达量的改变。与对照相比,NOKL220P表达量明显提高,而NOKL237P表达量降低。从NOK蛋白在细胞内的分布特征来看,不同双亮氨酸模体的突变同样会引起NOK蛋白在细胞内分布的改变,包括NOK蛋白形成聚集体的细胞比例、在细胞中的分布位置以及聚集体的大小和数量的不同。特别是NOKL203P、NOK237P以及L299P三个位置的突变可以显著地降低NOK蛋白在细胞中的聚集。不仅如此,过量表达NOKL41P还表现出了更为明显的核膜定位特征,而L220P则表现出了更为明显的质膜定位特征。由此说明,NOK的不同双亮氨酸模体对其蛋白质表达水平以及在细胞内的定位分布具有重要的调节作用,该研究为阐明NOK蛋白促肿瘤形成及转移的分子机理提供了新的见解。

NOK/STYK1 is a structurally unique member in receptor tyrosine kinase family （RTKs）, which has strong ability to facilitate tumorigenesis and metastasis. It is also involved in cellular endocytosis. Analysis of NOK protein sequence revealed that there were several di-leucine motifs. Di-leucine motifs have been reported to play important roles in cellular endocytosis, vesicle trafficking, signaling sorting and so on. In the present study,a series of mutants of NOK di-leucine motifs were established and their protein expression and cellular distribution pattern were analyzed in HeLa cells. The results indicated that mutation in different NOK di-leucine motifs caused different change of NOK protein expression level. The protein expression level of NOKL220P mutant was higher than wild type NOK whereas NOKL237P was lower than wild type. As to the cellular distribution, some mutants altered the cellular distribution pattern of NOK protein, including the proportion of dot pattern （DP）-containing cells to aggregation pattern （AP）-containing cells as well as the localization, the size and the number of NOK aggregates. The mutants of NOKL203P, NOK237P and L299P significantly reduced the formation of NOK aggregates in cells. In addition to the above, overexpression of NOKL41 had more distinctive localization on nuclear envelop whereas overexpression of L220P had more distinctive localization on cytoplasma membrane. Taken together, di-leucine motifs of NOK mediate its protein expression level and cellular distribution. Therefore, this study provides some new insights into understanding the molecular mechanism underlying NOK＇s facilitation to tuomrigensis and metastasis.