本文關(guān)鍵詞： c-Abl FoxM1 穩(wěn)定性 轉(zhuǎn)錄功能 相互作用 磷酸化 細胞周期進程 基因轉(zhuǎn)錄調(diào)控　出處：《中國人民解放軍軍事醫(yī)學科學院》2017年博士論文　論文類型：學位論文

【摘要】：c-Abl屬于非受體酪氨酸激酶,是Abelson鼠白血病病毒原癌基因v-abl在人類上的同源基因。c-Abl廣泛分布于各組織細胞當中,主要通過結(jié)合并且磷酸化其底物蛋白發(fā)揮功能;c-Abl可參與細胞增殖、細胞分化、腫瘤發(fā)生形成、DNA損傷修復(fù)、細胞骨架生成、細胞凋亡、細胞氧化應(yīng)激等生理功能調(diào)控。c-Abl相關(guān)蛋白Arg,與c-Abl在結(jié)構(gòu)上高度同源,發(fā)揮相似的功能。FoxM1是一個主要存在于增殖分化細胞中的轉(zhuǎn)錄因子,屬于FOX蛋白家族,它通過調(diào)控基因的轉(zhuǎn)錄表達參與細胞增殖、細胞周期調(diào)控、DNA損傷修復(fù)、腫瘤發(fā)生形成、胚胎發(fā)育及組織器官再生、細胞凋亡等生理過程。由于兩者密切參與細胞周期進程、腫瘤發(fā)生形成、DNA損傷修復(fù)等,且先前已有報道,c-Abl能夠調(diào)控p53、NF-κB、C/EBPβ等轉(zhuǎn)錄因子,因此c-Abl對FoxM1的調(diào)控機制值得探討。我們前期的研究發(fā)現(xiàn),過表達的c-Abl能夠結(jié)合并且磷酸化FoxM1,我們通過質(zhì)譜分析(LC-MS/MS)鑒定出5個主要的磷酸化位點(分別為Y129、Y272、Y317、Y362、Y575)。本研究將對兩者的相互作用,c-Abl對FoxM1的磷酸化修飾進行更深入的探討,并著重研究c-Abl調(diào)控FoxM1的分子機制和生理意義,包括泛素化調(diào)控、表達水平調(diào)控和轉(zhuǎn)錄功能調(diào)控,以及這些調(diào)控作用對細胞周期進程和細胞凋亡的影響。在本研究中,我們首先使用免疫共沉淀、免疫印跡和激光共聚焦等技術(shù)手段,證實內(nèi)源性的c-Abl與FoxM1存在相互作用,相互作用主要定位于細胞核,且主要發(fā)生于有絲分裂期(M期)。進一步,我們證實內(nèi)源性FoxM1可被酪氨酸磷酸化修飾,并且c-Abl激酶特異性抑制劑STI571能夠顯著抑制FoxM1酪氨酸磷酸化。接下來,我們研究c-Abl對FoxM1表達水平的影響,發(fā)現(xiàn)c-abl/arg被敲低或敲除后,FoxM1的表達水平也出現(xiàn)明顯下調(diào);同時發(fā)現(xiàn),使用STI571抑制細胞中的c-Abl激酶活性,也能夠顯著下調(diào)FoxM1的表達水平,說明FoxM1的表達依賴于c-Abl激酶活性。我們使用放線菌酮(CHX)處理細胞,抑制蛋白質(zhì)合成,檢測MEF WT細胞和MEF(c-abl-/-/arg-/-)細胞中FoxM1蛋白的半衰期,發(fā)現(xiàn)c-abl/arg敲除后,FoxM1的半衰期顯著縮短,說明c-Abl/Arg能夠增強FoxM1的蛋白穩(wěn)定性。我們猜測,c-Abl對FoxM1的磷酸化調(diào)控其經(jīng)泛素--蛋白酶體途徑降解,導(dǎo)致其表達水平受到影響。為此,通過使用STI571和蛋白酶體抑制劑MG132處理U2OS細胞,檢測FoxM1的泛素化程度和蛋白表達水平,發(fā)現(xiàn)STI571處理顯著增強其泛素化程度并下調(diào)其表達水平;而MG132處理能夠使其表達水平明顯回升,說明c-Abl能夠抑制FoxM1的泛素-蛋白酶體降解途徑,從而提高FoxM1的表達水平。進一步,我們對FoxM1磷酸化突變體的泛素化修飾和表達水平進行鑒定,發(fā)現(xiàn)與野生型FoxM1相比較,Y272F、Y575F及Y272/575F突變體的泛素化程度顯著增強,表達水平明顯下調(diào),同時與E3泛素連接酶組件CDH1的結(jié)合作用顯著增強,說明c-Abl對Y272和Y575這兩個位點的磷酸化,對FoxM1的穩(wěn)定性有重要作用。同時發(fā)現(xiàn)Y317F、Y362F突變體的表達水平明顯升高,并且CDH1與它們的結(jié)合作用顯著減弱,提示c-Abl對Y317、Y362的磷酸化可能促進FoxM1蛋白的降解。FoxM1通過調(diào)節(jié)大量的關(guān)鍵基因參與細胞周期調(diào)控,這些基因蛋白包括Cyclin B1、Cyclin D1、CDC25A、Plk1、CENP-F、Aurora-A及Aurora-B等。既然c-Abl能夠磷酸化并調(diào)控FoxM1表達水平,那么對FoxM1轉(zhuǎn)錄功能的影響也值得探討。我們發(fā)現(xiàn),當abl/arg被敲低后,細胞中的FoxM1及其下游靶基因蛋白Cyclin B1、Cyclin G2、Plk1的表達水平顯著下調(diào)。同時,使用STI571抑制c-Abl的激酶活性,也能夠顯著下調(diào)Cyclin B1、Plk1和CENP-F等的轉(zhuǎn)錄水平和表達水平,說明c-Abl對FoxM1的調(diào)控能夠顯著影響其轉(zhuǎn)錄功能。進一步,我們探討c-Abl對FoxM1的調(diào)控能否影響細胞周期進程。通過使用細胞周期同步化和流式細胞儀檢測等技術(shù)方法,我們證實STI 571通過抑制c-Abl激酶活性,下調(diào)FoxM1的表達水平,并抑制其轉(zhuǎn)錄功能,進而抑制其靶基因蛋白Cyclin B1、Plk1和Aurora-B等的轉(zhuǎn)錄表達,最終導(dǎo)致細胞周期阻滯。FoxM1的另一個重要功能是參與腫瘤細胞的抗凋亡,為此,我們研究c-Abl對FoxM1的調(diào)控是否影響腫瘤細胞的凋亡。通過使用STI571和阿霉素(Doxorubicin,能夠誘導(dǎo)腫瘤細胞凋亡)處理Hela細胞,檢測凋亡蛋白Cleaved caspase3和Cleaved PARP-1的表達水平,發(fā)現(xiàn)STI571處理加劇了阿霉素誘導(dǎo)的Hela細胞凋亡,說明STI571通過抑制c-Abl激酶活性,下調(diào)FoxM1表達水平,抑制FoxM1的抗凋亡功能。我們的研究證實,c-Abl能夠明顯調(diào)控轉(zhuǎn)錄因子FoxM1,并且根據(jù)文獻報道,c-Abl也能夠調(diào)控其他的轉(zhuǎn)錄因子如c-Myc、NF-κB、C/EBPβ等。因此我們猜測,c-Abl/Arg能夠參與調(diào)控基因的轉(zhuǎn)錄表達。為此,我們使用高通量測序(RNA-seq)和生物信息學分析的方法,對MCF-7WT及MCF-7 KD(c-abl/arg Knock-Down)細胞的RNA進行轉(zhuǎn)錄組分析。發(fā)現(xiàn)共有1034個基因的轉(zhuǎn)錄水平受到c-Abl/Arg的顯著調(diào)控,c-abl/arg敲低之后,有635個基因的轉(zhuǎn)錄水平下調(diào),399個基因的轉(zhuǎn)錄水平上調(diào),說明c-Abl/Arg能夠調(diào)控基因的轉(zhuǎn)錄水平,并且以正向調(diào)控為主。進一步,我們對受c-Abl/Arg調(diào)控的基因進行功能聚類分析,發(fā)現(xiàn)它們的功能參與多種信號通路和細胞代謝進程,并且這些通路和進程大多與已報道的c-Abl功能密切相關(guān),提示c-Abl不僅在蛋白水平對相關(guān)功能通路進行調(diào)控,并且在基因轉(zhuǎn)錄水平也參與調(diào)控。綜合以上研究結(jié)果,我們證實細胞內(nèi)源性的c-Abl能夠結(jié)合并磷酸化修飾FoxM1;c-Abl對FoxM1的磷酸化作用,抑制其經(jīng)泛素-蛋白酶體途徑降解,增強其穩(wěn)定性,上調(diào)其表達水平;c-Abl對FoxM1的調(diào)控影響細胞周期進程和腫瘤細胞凋亡;c-Abl/Arg能夠參與基因轉(zhuǎn)錄水平調(diào)控。本研究為深入探討c-Abl和FoxM1的功能,特別是探討兩者在細胞周期進程和腫瘤細胞凋亡等方面的功能,提供重要的理論支撐。
[Abstract]:C-Abl belongs to the non receptor tyrosine kinase, Abelson murine leukemia virus oncogene v-abl in human homologous gene.C-Abl is widely distributed in various tissues and cells, mainly through the combination and phosphorylation of substrate proteins; c-Abl may be involved in cell proliferation, cell differentiation, tumor formation, DNA damage repair, cell skeleton generation. Cell apoptosis, oxidative stress physiological functions such as regulation of.C-Abl related protein Arg, and c-Abl is highly homologous in structure, function similar to.FoxM1 is a transcription factor mainly exists in the proliferation and differentiation of cells, which belongs to the FOX protein family, its expression by transcriptional regulation of genes involved in cell proliferation, cell cycle regulation, DNA damage repair, tumor formation, embryonic development and tissue regeneration, apoptosis and other physiological processes. Because the two are closely involved in the process of cell cycle, tumor The formation and repair of DNA damage, and have been reported previously, c-Abl can regulate p53, NF- kappa B, C/EBP beta and other transcription factors, therefore the regulation mechanism of c-Abl on FoxM1 is worth exploring. Our previous study showed that overexpression of c-Abl can bind and phosphorylation of FoxM1, we through mass spectrum analysis (LC-MS/MS) identified 5 major phosphorylation sites (Y129, Y272, Y317, Y362, Y575). The interaction of the two, phosphorylation of FoxM1 c-Abl to further discuss the molecular mechanism and physiological significance, and focuses on the regulation of FoxM1 c-Abl, including ubiquitin regulation, expression regulation the function and transcriptional regulation, and the influence of these effects on cell cycle progression and cell apoptosis. In this study, we used immunoprecipitation, immunoblotting and confocal technique, c-Abl and FoxM1 confirmed that endogenous. In the interaction, the interaction was mainly located in the nucleus, and occurs mainly in the mitotic phase (M phase). Further, we demonstrate that endogenous FoxM1 may be tyrosine phosphorylation, and c-Abl kinase specific inhibitor STI571 can significantly inhibit the tyrosine phosphorylation of FoxM1. Next, we study the effects of c-Abl on the expression of FoxM1. C-abl/arg was found to knockdown or knockout, expression of FoxM1 was significantly reduced; at the same time, the use of STI571 inhibition of c-Abl kinase activity in cells, can also down-regulation of FoxM1, indicating c-Abl dependent kinase activity and the expression of FoxM1. We use the cycloheximide treated cells, inhibition (CHX) protein synthesis, detection of MEF WT cells and MEF (c-abl-/-/arg-/-) FoxM1 protein half-life, found that c-abl/arg knockout, the half-life of FoxM1 was significantly reduced, indicating that c-Abl/Arg can enhance The stability of FoxM1 protein. We speculate that phosphorylation of FoxM1 c-Abl by the ubiquitin - proteasome pathway leads to degradation, its expression level is affected. Therefore, by using STI571 and proteasome inhibitor MG132 treated U2OS cells, detect the ubiquitination of FoxM1 and protein expression level, found that STI571 treatment significantly enhanced its ubiquitin the degree and down-regulation of its expression level; while MG132 treatment can make the expression level increase, indicating that c-Abl can inhibit FoxM1 degradation by the ubiquitin proteasome pathway, so as to improve the expression level of FoxM1. Further, we on the phosphorylation of FoxM1 mutant ubiquitination and identify the expression level of FoxM1, compared with the wild type Y272F, Y575F and Y272/575F, the degree of ubiquitination of mutant significantly enhanced expression levels were significantly reduced, at the same time node cooperation with E3 ubiquitin ligase CDH1 components with significant C-Abl enhanced on Y272 and Y575 of the two phosphorylation plays an important role on the stability of FoxM1. At the same time that Y317F, the expression level of Y362F mutant was significantly increased, and the combination of CDH1 and their reduced significantly, suggesting that c-Abl of Y317, the phosphorylation of Y362 could promote the degradation of.FoxM1 protein by FoxM1 a large number of regulating key genes involved in cell cycle regulation, these proteins including Cyclin B1, Cyclin D1, CDC25A, Plk1, CENP-F, Aurora-A and Aurora-B. Since c-Abl can phosphorylate and regulate the expression level of FoxM1, then the influence on the transcriptional function of FoxM1 is also worth exploring. We found that, when knockdown of abl/arg cells the FoxM1 and its downstream target gene Cyclin B1, Cyclin G2, the expression level of Plk1 is significantly decreased. At the same time, the use of STI571 to inhibit c-Abl kinase activity, can significantly reduce the Cyclin of B1, Plk1 and CENP-F etc. The transcription and expression level, c-Abl regulation of FoxM1 can significantly affect the transcriptional function. Further, we investigate the c-Abl regulation of FoxM1 can influence the cell cycle progression. Through the use of cell cycle synchronization and flow cytometry technique, we confirmed that STI 571 through inhibition of c-Abl kinase activity and down-regulation of expression level FoxM1, and inhibit its transcriptional function, thereby inhibiting its target gene Cyclin B1 protein, expression of Plk1 and Aurora-B, resulting in another important function of.FoxM1 cell cycle arrest is involved in tumor cell apoptosis, therefore, we study the c-Abl regulation of FoxM1 will affect the apoptosis of tumor cells through the use of. (Doxorubicin, STI571 and adriamycin could induce the apoptosis of tumor cells) treated Hela cells, apoptosis protein Cleaved Caspase3 and Cleaved PARP-1 expression was found. STI571 treatment increased Hela cell apoptosis induced by doxorubicin, STI571 through inhibition of c-Abl kinase activity, decrease the expression level of FoxM1 and inhibition of FoxM1 anti apoptotic function. Our study confirmed that c-Abl can significantly regulate the transcription factor FoxM1, and according to the literature, c-Abl can regulate the transcription of other factors such as c-Myc, NF- K B. C/EBP beta. So we speculated that the expression of c-Abl/Arg can participate in the regulation of genes. Therefore, we used high-throughput sequencing (RNA-seq) analysis and bioinformatics, MCF-7WT and MCF-7 (KD c-abl/arg Knock-Down) RNA cell transcriptome analysis. There were significant transcriptional regulation of 1034 genes by c-Abl/Arg, c-abl/arg knockdown, transcription levels of 635 genes downregulated, 399 genes transcription level increased, indicating that c-Abl/Arg can control the gene transcription level, and In the positive control. Further, we performed functional clustering analysis of genes regulated by c-Abl/Arg, found that their functions involved in various signaling pathways and cellular metabolic processes, and most of these pathways and processes with the reported c-Abl function closely related, suggesting that c-Abl not only at the protein level of related functional regulatory pathways, and also participate in the level of gene transcription regulation. Based on the above results, we demonstrate that endogenous c-Abl binding and phosphorylation of FoxM1; phosphorylation of c-Abl on FoxM1, the inhibition by ubiquitin proteasome degradation pathway, enhance its stability, increase its expression level; c-Abl regulation effect on FoxM1 cell cycle progression and the apoptosis of tumor cells; c-Abl/Arg can participate in gene transcription regulation. This study to further explore the function of FoxM1 and c-Abl, especially on the cell cycle It provides important theoretical support for the function of stage and tumor cell apoptosis.

【學位授予單位】：中國人民解放軍軍事醫(yī)學科學院
【學位級別】：博士
【學位授予年份】：2017
【分類號】：Q78

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