CK1介导磷酸化调控OREBP低渗透压条件下的核浆转运

发布时间：2018-04-24 13:25

本文选题：OREBP + CK1　；参考：《重庆医科大学》2008年博士论文

【摘要】： 渗透压反应增强子结合蛋白(Osmolarity Response Enhancer Binding Protein,OREBP或称Tonicity Enhancer Binding Protein,TonEBP),又名:活化T淋巴细胞核因子5 (Nuclear Factor of Activated T Cell 5,NFAT5)是迄今为止唯一已知的哺乳动物细胞渗透压反应调节因子。当细胞外微环境渗透压升高时,OREBP作为渗透压相关的转录因子,从细胞浆进入细胞核,结合到一系列渗透压相关靶基因上游的渗透压反应增强子区域(Osmolarity Response Enhancer,ORE),启动它们的表达,提高细胞内相应渗透性溶质浓度,从而降低胞内离子浓度,维持细胞正常生理功能。当细胞外渗透压降低,OREBP从胞核转运到胞浆,相关基因的转录水平随之降低。生理情况下,肾脏髓质,软骨及消化道上皮等组织器官所处环境的渗透压波动极大,持续时间长,渗透压反应机制是维持这些组织器官正常生理功能的最重要防御机制。近来,细胞核、浆之间的运动已成为OREBP功能研究的热点,其具体机制有一定的发现,但是仍然不够详尽。本课题组于2006年发现了三个控制OREBP核浆转运的结构域:NLS(Nuclear Localization Signal),NES(Nuclear Export Signal)和AED(Auxiliary Export Domain,AED),NLS负责OREBP高渗、等渗下入细胞核,NES通过与CRM1(Nuclear Export Receptor Exportin 1)作用负责其等渗、低渗下出细胞核,而AED(132-156aa)可在低渗条件下,独自携带OREBP出细胞核,这一过程不依赖于NES,那么AED是通过什么机制介导OREBP出胞核的呢?最近的研究发现:磷酸化修饰能影响蛋白质的分子构象及其与核浆转运蛋白的相互作用,从而影响该蛋白的核浆转运过程。低渗刺激是否通过磷酸化AED区域的某些氨基酸残基,改变OREBP的构象,促使其出细胞核?本课题围绕这一问题展开了相关研究。 1.使用丙氨酸点突变筛查方法,将AED区域9个候选氨基酸逐个突变成惰性丙氨酸,观察突变后FLAG-OREBP_(1-581)Δ1-131融合蛋白在低渗刺激下的亚细胞定位。我们发现:野生型融合蛋白:胞核型细胞占9%,胞浆型细胞占79%;S155A突变具有明显抑制低渗出核效果,胞核型细胞占66%,胞浆型细胞占16.7%,提示:S155位点在低渗出胞核过程中有重要作用。 2.低渗处理后,对FLAG-OREBP_(1-581)进行质谱检测发现,S155及S158两个位点发生磷酸化修饰。将这两个位点突变为丙氨酸或者天门冬氨酸的实验发现, FLAG-OREBP_(1-581)Δ1-131S155A、S158A及S155/158A突变融合蛋白均不能在低渗下出胞核,S155D突变融合蛋白能顺利出胞核,而S158D及S155/158D两个突变融合蛋白低渗出胞核受抑。同时我们构建GFP-OREBP_(1-581)融合蛋白及各个突变体,使用激光共聚焦显微镜实时观察低渗刺激下的GFP融合蛋白出胞核进程,结果与细胞免疫化学分析一致。因此我们认为:只有S155和S158按先后顺序依次发生磷酸化修饰,OREBP才能在低渗刺激下顺利出胞核。 3.使用GST-OREBP146-167融合蛋白及各个突变体作为底物,进行体外激酶活性实验,发现细胞核内有未知激酶在低渗刺激下迅速激活,S158位点磷酸化以S155位点磷酸化为前提,在体内使用FLAG-OREBP_(1-581)Δ1-131及各个突变体,通过凝胶电泳滞后实验证实了体外激酶实验的结果。另一方面,根据生物信息学分析提示信息,采用重组人CK1α1,证实当S155磷酸化后,S158位点成为了CK1的作用靶点,CKI-7抑制低渗刺激胞核提取物内CK1活性后,GST-OREBP146-167S155D的磷酸化修饰明显受抑制;使用siRNA抑制体内CK1各亚型表达,发现CK1α1L在OREBP低渗刺激下的出胞核过程中发挥重要作用,同时使用CK1α1L shRNA进一步证实实验结果。通过以上实验,我们获得以下结论: 低渗刺激下胞核内一个未知的激酶迅速活化,磷酸化OREBP AED区域的S155位点,紧接着CK1α1L磷酸化S158位点,此两个位点发生磷酸化修饰后,OREBP才能从细胞核顺利进入细胞浆。
[Abstract]:The osmotic reaction enhancer binding protein (Osmolarity Response Enhancer Binding Protein, OREBP or Tonicity Enhancer Binding Protein, TonEBP), also known as the activated T lymphocyte nuclear factor 5, is the only known regulation factor for the osmotic pressure of mammalian cells so far. When the osmotic pressure of the extracellular microenvironment is high, OREBP is a transcription factor associated with osmotic pressure, from the cytoplasm into the nucleus and binding to a series of osmotic pressure response enhancers (Osmolarity Response Enhancer, ORE) upstream of the osmotic pressure related target genes, starting their expression, improving the intracellular concentration of the corresponding osmotic solute, and thus reducing the concentration of the corresponding osmotic solute in the cell The concentrations of intracellular ions maintain normal physiological functions of the cells. When the osmotic pressure decreases, the OREBP transshipment from the nucleus to the cytoplasm, the transcriptional level of the related genes decreases. Under physiological conditions, the osmotic pressure of the tissues and organs such as the medulla, cartilage and the digestive tract of the kidneys is greatly increased, the duration of the osmotic pressure is long, and the osmotic pressure mechanism is maintained. The most important defense mechanisms of these tissues and organs are normal physiological functions.
Recently, the movement between the nucleus and the plasma has become a hot spot in the research of OREBP function, and its specific mechanism has been found, but it is still not exhaustive. In 2006, we found three domains that control the OREBP nuclear plasma transport: NLS (Nuclear Localization Signal), NES (Nuclear Export Signal) and AED (Auxiliary). LS is responsible for OREBP hyperosmotic, isosmotic into the nucleus, NES is responsible for its isosmotic and hypotonic nucleus through the action of CRM1 (Nuclear Export Receptor Exportin 1), and AED (132-156aa) can carry the OREBP nucleus of the nucleus under the hypotonic condition, this process is not dependent on NES, then what mechanism has been used to mediate the nucleus? The study found that phosphorylation can affect the molecular conformation of protein and its interaction with the nucleoplasma transporter, which affects the nucleoplasma transport process of the protein. Whether or not the hypotonic stimulation changes the conformation of OREBP through some amino acid residues in the phosphorylated AED region, and promotes its nucleus out of the nucleus. Research.
1. using the alanine point mutation screening method, 9 candidate amino acids in the AED region were mutated to inert alanine one by one, and the subcellular localization of FLAG-OREBP_ (1-581) Delta 1-131 fusion protein was observed under the hypotonic stimulation. We found that the wild type fusion protein was 9%, the cytoplasm cells accounted for 79%, and the S155A mutation was obviously inhibited. The effect of low exudate nucleus was 66% and cytoplasmic cells accounted for 16.7%, suggesting that the S155 locus plays an important role in the process of low exudate nucleus.
After 2. hypotonic treatment, FLAG-OREBP_ (1-581) was detected by mass spectrometry, and two sites of S155 and S158 were phosphorylated. The two sites were mutated to alanine or aspartic acid, and the FLAG-OREBP_ (1-581) Delta 1-131S155A, S158A and S155/158A mutant fusion proteins were not low permeable nuclei and S155D mutation fusion. The protein can smooth out the nucleus, while the low exudation nuclei of the two fusion proteins of S158D and S155/158D are suppressed. We construct the GFP-OREBP_ (1-581) fusion protein and the various mutants. The process of the GFP fusion protein exocytosis under the hypotonic stimulation is observed by laser confocal microscopy. The result is consistent with the cell immunocytochemical analysis. We believe that only S155 and S158 can undergo phosphorylation in sequence, and OREBP can smooth out the nucleus under hypotonic stimulation.
3. using the GST-OREBP146-167 fusion protein and the various mutants as substrates, the activity of kinase activity in vitro was carried out. It was found that the unknown kinase in the nucleus was activated rapidly under the hypotonic stimulation. The phosphorylation of the S158 site was based on the phosphorylation of the S155 site, and the FLAG-OREBP_ (1-581) Delta 1-131 and the various mutants were used in the body, and the gel electrophoresis was delayed. On the other hand, on the other hand, according to the information of bioinformatics analysis, the recombinant human CK1 alpha 1 was used to confirm that when S155 phosphorylation, the S158 site became the target of CK1, and the phosphorylation of GST-OREBP146-167S155D was obviously inhibited after CKI-7 inhibited the CK1 activity in the nucleus extracts of the hypotonic stimulation; and siRNA was used. Inhibiting the expression of CK1 subtypes in vivo, it is found that CK1 alpha 1L plays an important role in the process of nucleus exocytosis under OREBP hypotonic stimulation. Meanwhile, CK1 alpha 1L shRNA is used to further confirm the results of the experiment.
An unknown kinase in the nucleus of the cell is activated rapidly under the hypotonic stimulation, and the S155 site of the phosphorylated OREBP AED region is followed by the CK1 alpha 1L phosphorylated S158 site. After phosphorylation of the two sites, the OREBP can enter the cytoplasm smoothly from the nucleus.

【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R341

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