人类RPK118基因的功能研究

发布时间：2018-05-17 03:30

本文选题：RPK118 + PRDX3　；参考：《复旦大学》2005年博士论文

【摘要】：由于发现我室1999年报道的RPS6KC1基因在肝癌组织中上调表达,我们拟对其生理功能进行详细研究,但在核对测序数据时却发现其cDNA序列并非是全长序列,于是通过EST电子信息步移的方法,在GenBank数据库中检索到了若干位于5’侧的新ESTs序列,经重新拼接得到的全长cDNA序列长达4196bp,较之原序列(2349bp)长了1847bp,与原先Northem杂交检测到的转录本长度4.4kb非常接近。新序列的开放阅读框为3102bp,它编码了1066个氨基酸,预测蛋白分子量为118.7KD。用全长cDNA检索人类基因组序列发现,该基因由15个外显子和14个内含子组成,位于染色体1q32.3,我们从人脑cDNA文库中克隆到了全长的RPS6KC1 cDNA,测序证明所克隆的cDNA与来自ESTS拼接的序列完全一致。经用SMART软件作RPS6KC1功能结构域分析,发现预测的RPS6KC1具有4个结构域,依次它们分别是PX结构域(PX domain)、MIT结构域(Microtubule-Interacting and Trafficking moleculars domain)、STK1和STK2结构域(Serine/Threonine protein kinases,catalytic domain)。为了证明RPS6KC1蛋白的客观存在并为进一步研究该蛋白的功能提供材料,我们在原核细胞中重组表达了RPS6KC1表位抗原区的肽段,并用其制备了RPS6KC1的多克隆抗体。Westem Blot证实RPS6KC1确实存在于细胞中,其分子量约为120KD。基于预测的RPS6KC1具有间隔分布的蛋白激酶结构域,我们又用哺乳动物细胞表达了RPS6KC1的全蛋白,并以MBP(Myline Binding Protein)以及S6肽段为底物,检测了RPS6KC1的蛋白激酶活性,但未能获得阳性结果。由于这一实验结果与日本学者在同期获得的研究结果一致,我们采纳了日本学者对这个基因的命名(2002年9月发表于JBC),RPK118(既118KD的核糖体假激酶)。虽然RPK118蛋白没有激酶活性,但日本学者发现它能够与鞘氨醇激酶互作,并且可共定位于内涵体上,非常巧合的是我们用酵母双杂交、Pull-Down和免疫共沉淀等方法发现PRK118能够与一个已知定位于线粒体的抗氧化蛋白PRDX3互作,并且它也能把PRDX3共定位到内涵体上。在293T和COS7细胞中,荧光抗体标记的RPK118弥散分布、或者成小环状、颗粒状分布于细胞质中,与早期内涵体标志蛋白EEA1呈现共定位图像。在RPK118与PRDX3共转染的细胞中,RPK118的分布与单独转染时相同,而PRDX3与EEA1呈现明确的共定位图像,这在单独转染PRDX3时很难观察到。然而,当用缺失N端PX区的RPK118突变体与PRDX3共转染时,二者显现共定位图像,但都不能与EEA1共定位。当用缺失C端激酶区的RPK118突变体与PRDX3共转染时,则仅见RPK118与EEA1的共定位,观察不到PRDX3与EEA1的共定位。这些结果提示,RPK118的PX结构域是参与内涵体定位的区域,而其激酶结构域可能是PRDX3的
[Abstract]:Due to the discovery of the up-regulation of RPS6KC1 gene expression in HCC tissues reported in our laboratory in 1999, we intend to study its physiological function in detail, but when we check the sequencing data, we find that its cDNA sequence is not a full-length sequence. A number of new ESTs sequences located on the 5'side were retrieved in the GenBank database by the method of EST electronic information step. The full-length cDNA sequence was 4196bp, 1847 BP longer than the original sequence 2349bp, which was very close to the length 4.4kb detected by Northem hybridization. The open reading frame of the new sequence is 3102 BP, which encodes 1066 amino acids, and the predicted molecular weight of the protein is 118.7 KD. Searching the human genome sequence by full-length cDNA, we found that the gene consists of 15 exons and 14 introns. At chromosome 1q32.3. the full-length RPS6KC1 cDNA was cloned from the human brain cDNA library. The sequence of the cloned cDNA was identical to the sequence from the ESTS splicing. By using SMART software to analyze the functional domain of RPS6KC1, it is found that the predicted RPS6KC1 has four domains, which are PX domain PX domain, Microtubule-Interacting and Trafficking moleculars domain STK1 and STK2 domain Serine / Threonine protein kinasescattic domain respectively, which are respectively microtubule-interacting and Trafficking moleculars domain STK1 and STK2 domain Serine / Threonine protein kinasescatalytic domain. In order to prove the existence of RPS6KC1 protein and provide materials for further study of its function, we expressed the peptide of RPS6KC1 epitope in prokaryotic cells. The polyclonal antibody of RPS6KC1. Westem Blot showed that RPS6KC1 existed in the cells, and its molecular weight was about 120kD. Based on the predicted protein kinase domain of RPS6KC1, we expressed the whole protein of RPS6KC1 in mammalian cells and detected the protein kinase activity of RPS6KC1 using MBP(Myline Binding protein and S6 peptide as substrates, but no positive results were obtained. As the results of this study are consistent with those obtained by Japanese scholars in the same period, we have adopted the name of this gene by Japanese scholars (published in September 2002 in JBCX RPK118, the ribosomal pseudokinase of 118KD. Although the RPK118 protein has no kinase activity, Japanese researchers have found that it can interact with sphingosine kinase and co-locate on the connotations. Coincidentally, we found that PRK118 can interact with an antioxidant protein known to be located in mitochondria, PRDX3, by yeast two-hybrid pull-down and immunoprecipitation, and it can also co-locate PRDX3 on the connotative body. In 293T and COS7 cells, fluorescent antibody labeled RPK118 was dispersed, or formed a small circular, granular distribution in the cytoplasm, showing a co-localization image with the early connotative marker protein EEA1. The distribution of RPK118 in the co-transfected cells of RPK118 and PRDX3 was the same as that in the single transfection, while PRDX3 and EEA1 showed a clear co-localization image, which was difficult to observe when PRDX3 was transfected alone. However, when the RPK118 mutants with missing N-terminal PX region were co-transfected with PRDX3, the co-localization images were displayed, but neither of them could be co-located with EEA1. When the RPK118 mutant with missing C-terminal kinase region was co-transfected with PRDX3, only the co-localization of RPK118 and EEA1 was observed, but the co-localization of PRDX3 and EEA1 was not observed. These results suggest that the PX domain of RPK118 is the domain involved in the localization of intension, and its kinase domain may be PRDX3's.
【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2005
【分类号】：R346;Q987

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