NFAR1稳定IL-2 mRNA上游调节机制的研究

发布时间：2018-03-29 17:09

本文选题：NFAR1　切入点：AKT　出处：《复旦大学》2006年博士论文

【摘要】： CD28所诱导的白细胞介素(IL-2)的转录后调控被认为是与IL-2 mRNA3'UTR区的富集腺嘌呤尿嘧啶保守区域(AU-rich elements,AREs)相关的。与ARE结合的蛋白(AU-binding protein,AUBP)能调节mRNA去腺苷酸,改变mRNA降解的速率,磷酸化AUBPs可以稳定mRNA。NFAR1是一个与IL-2 mRNAARE区结合的AUBP,在过量表达时可以稳定IL-2 mRNA。NFAR1基因编码区含有一个保守的AKT底物序列,其中S647位是AKT保守的磷酸化位点。在本文中证实了AKT与NFAR1相互作用的,并共定位于细胞核,并用体外实验和体内实验证明了AKT确实可以磷酸化NFAR1的S 647位。为了进一步说明AKT磷酸化NFAR1的生理意义,我们检测了外源表达NFAR1野生型,失磷酸化突变型NFAR1-S647A及模拟S647被磷酸化的假磷酸化突变型NFAR1-S647E对IL-2 mRNA半衰期的影响。其中野生型可以稳定IL-2mRNA,NFAR1-S647A则和空载体CMV-Myc类似,稳定IL-2 mRNA能力很低;而转染NFAR1-S647E的细胞中IL-2 mRNA稳定性与野生型近似,略高于野生型。我们还观察到AKT和野生型NFAR1共转的Jurkat细胞中IL-2mRNA稳定性要远远高于AKT和NFAR1S647A共转的细胞,也高于单转NFAR1的细胞;P13-K通路的抑制剂LY290042抑制NFAR1野生型稳定IL-2 mRNA的功能,NFAR1-S647A则对LY290042不敏感。通过上述实验证明了AKT通过磷酸化NFAR1 S647调节IL-2mRNA的稳定性。我们进一步检测CD28刺激信号对NFAR1磷酸化程度的影响以及NFAR1S647位的磷酸化对CD28所介导的IL-2转录后调控所起的作用。发现CD28刺激后,NFAR1的磷酸化水平明显增加,而且这种增加是伴随着ART磷酸化水平增加的;一旦AKT被抑制,S647位的磷酸化水平也随之下降。放射免疫方法检测到NFAR1野生型和NFAR1-S647A对CD28所诱导的转录后调控有明显的不同。NFAR1可以稳定IL-2 mRNA并提高蛋白表达量,突变型则没有这样的作用,这说明NFAR1S647位的磷酸化对CD28稳定IL-2 mRNA非常重要。根据上述的研究结果,我们提出了一条CD28稳定IL-2 mRNA可能的分子通路;CD28刺激激活AKT,活化的AKT进入细胞核内磷酸化NFAR1 S647位,促使IL-2 mRNA稳定性增加。
[Abstract]:The posttranscriptional regulation of interleukin 2 (IL 2) induced by CD28 is thought to be related to the AU-rich elements ARES (a conserved region of IL-2 mRNA3'UTR). The AU-binding protein (AU-binding protein) associated with ARE can regulate adenylate in mRNA and alter the degradation rate of mRNA. Phosphorylated AUBPs can stabilize mRNA.NFAR1, which binds to IL-2 mRNAARE region, and when overexpressed, IL-2 mRNA.NFAR1 gene coding region contains a conserved AKT substrate sequence. S647 is the conserved phosphorylation site of AKT. In this paper, the interaction between AKT and NFAR1 is confirmed and co-located in nucleus. In vitro and in vivo experiments, it is proved that AKT can indeed phosphorylate S647 of NFAR1. In order to elucidate the physiological significance of AKT phosphorylated NFAR1, we detected the wild type of NFAR1 expression. The effects of dephosphorylation mutant NFAR1-S647A and pseudophosphorylation mutant NFAR1-S647E mimicking S647 phosphorylation on the half-life of IL-2 mRNA. The wild type can stabilize IL-2mRNA-NFar1-S647A, which is similar to the empty vector CMV-Myc, and the ability of stabilizing IL-2 mRNA is very low. However, the stability of IL-2 mRNA in NFAR1-S647E transfected cells was similar to that of wild type and was slightly higher than that of wild type. We also observed that the IL-2mRNA stability of Jurkat cells cotransfected with AKT and wild type NFAR1 was much higher than that of AKT and NFAR1S647A cotransfected Jurkat cells. The inhibitory effect of LY290042, an inhibitor of P13-K pathway on NFAR1 wild-type stable IL-2 mRNA, was also higher than that of mono-transformed NFAR1. NFar1-S647A was not sensitive to LY290042. It was proved that AKT regulates the stability of IL-2mRNA by phosphorylation of NFAR1 S647. We further examined the effect of CD28 stimulation signal on NFAR1 phosphorylation and the effect of NFAR1S647 site phosphorylation on IL-2 post-transcriptional regulation mediated by CD28. We found that the phosphorylation level of CD28 stimulated IL-2 was significantly increased. Moreover, this increase was accompanied by an increase in ART phosphorylation levels. Once AKT was inhibited, the phosphorylation level of S647 was also decreased. Radioimmunoassay showed that NFAR1 wild-type and NFAR1-S647A had significantly different post-transcriptional regulation induced by CD28. NFA-1 could stabilize IL-2 mRNA and increase protein expression. The mutant does not, which suggests that phosphorylation of NFAR1S647 site is important for CD28 to stabilize IL-2 mRNA. Based on the above results, we propose a possible molecular pathway of CD28 stable IL-2 mRNA: CD28 stimulates the activation of AKT and activates AKT into the nucleus of phosphorylated NFAR1 S647, which promotes the stability of IL-2 mRNA.
【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2006
【分类号】：R392

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