本文選題：NFAR1　切入點(diǎn)：AKT　出處：《復(fù)旦大學(xué)》2006年博士論文

【摘要】： CD28所誘導(dǎo)的白細(xì)胞介素(IL-2)的轉(zhuǎn)錄后調(diào)控被認(rèn)為是與IL-2 mRNA3'UTR區(qū)的富集腺嘌呤尿嘧啶保守區(qū)域(AU-rich elements,AREs)相關(guān)的。與ARE結(jié)合的蛋白(AU-binding protein,AUBP)能調(diào)節(jié)mRNA去腺苷酸,改變mRNA降解的速率,磷酸化AUBPs可以穩(wěn)定mRNA。NFAR1是一個(gè)與IL-2 mRNAARE區(qū)結(jié)合的AUBP,在過(guò)量表達(dá)時(shí)可以穩(wěn)定IL-2 mRNA。NFAR1基因編碼區(qū)含有一個(gè)保守的AKT底物序列,其中S647位是AKT保守的磷酸化位點(diǎn)。在本文中證實(shí)了AKT與NFAR1相互作用的,并共定位于細(xì)胞核,并用體外實(shí)驗(yàn)和體內(nèi)實(shí)驗(yàn)證明了AKT確實(shí)可以磷酸化NFAR1的S 647位。 為了進(jìn)一步說(shuō)明AKT磷酸化NFAR1的生理意義,我們檢測(cè)了外源表達(dá)NFAR1野生型,失磷酸化突變型NFAR1-S647A及模擬S647被磷酸化的假磷酸化突變型NFAR1-S647E對(duì)IL-2 mRNA半衰期的影響。其中野生型可以穩(wěn)定IL-2mRNA,NFAR1-S647A則和空載體CMV-Myc類(lèi)似,穩(wěn)定IL-2 mRNA能力很低;而轉(zhuǎn)染NFAR1-S647E的細(xì)胞中IL-2 mRNA穩(wěn)定性與野生型近似,略高于野生型。我們還觀(guān)察到AKT和野生型NFAR1共轉(zhuǎn)的Jurkat細(xì)胞中IL-2mRNA穩(wěn)定性要遠(yuǎn)遠(yuǎn)高于AKT和NFAR1S647A共轉(zhuǎn)的細(xì)胞,也高于單轉(zhuǎn)NFAR1的細(xì)胞;P13-K通路的抑制劑LY290042抑制NFAR1野生型穩(wěn)定IL-2 mRNA的功能,NFAR1-S647A則對(duì)LY290042不敏感。通過(guò)上述實(shí)驗(yàn)證明了AKT通過(guò)磷酸化NFAR1 S647調(diào)節(jié)IL-2mRNA的穩(wěn)定性。 我們進(jìn)一步檢測(cè)CD28刺激信號(hào)對(duì)NFAR1磷酸化程度的影響以及NFAR1S647位的磷酸化對(duì)CD28所介導(dǎo)的IL-2轉(zhuǎn)錄后調(diào)控所起的作用。發(fā)現(xiàn)CD28刺激后,NFAR1的磷酸化水平明顯增加,而且這種增加是伴隨著ART磷酸化水平增加的;一旦AKT被抑制,S647位的磷酸化水平也隨之下降。放射免疫方法檢測(cè)到NFAR1野生型和NFAR1-S647A對(duì)CD28所誘導(dǎo)的轉(zhuǎn)錄后調(diào)控有明顯的不同。NFAR1可以穩(wěn)定IL-2 mRNA并提高蛋白表達(dá)量,突變型則沒(méi)有這樣的作用,這說(shuō)明NFAR1S647位的磷酸化對(duì)CD28穩(wěn)定IL-2 mRNA非常重要。 根據(jù)上述的研究結(jié)果,我們提出了一條CD28穩(wěn)定IL-2 mRNA可能的分子通路;CD28刺激激活A(yù)KT,活化的AKT進(jìn)入細(xì)胞核內(nèi)磷酸化NFAR1 S647位,促使IL-2 mRNA穩(wěn)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.
【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2006
【分類(lèi)號(hào)】：R392

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