c-Abl调控FoxM1稳定性及其转录功能研究

发布时间：2018-01-29 01:05

本文关键词： c-Abl FoxM1 稳定性转录功能相互作用磷酸化细胞周期进程基因转录调控　出处：《中国人民解放军军事医学科学院》2017年博士论文　论文类型：学位论文

【摘要】：c-Abl属于非受体酪氨酸激酶,是Abelson鼠白血病病毒原癌基因v-abl在人类上的同源基因。c-Abl广泛分布于各组织细胞当中,主要通过结合并且磷酸化其底物蛋白发挥功能;c-Abl可参与细胞增殖、细胞分化、肿瘤发生形成、DNA损伤修复、细胞骨架生成、细胞凋亡、细胞氧化应激等生理功能调控。c-Abl相关蛋白Arg,与c-Abl在结构上高度同源,发挥相似的功能。FoxM1是一个主要存在于增殖分化细胞中的转录因子,属于FOX蛋白家族,它通过调控基因的转录表达参与细胞增殖、细胞周期调控、DNA损伤修复、肿瘤发生形成、胚胎发育及组织器官再生、细胞凋亡等生理过程。由于两者密切参与细胞周期进程、肿瘤发生形成、DNA损伤修复等,且先前已有报道,c-Abl能够调控p53、NF-κB、C/EBPβ等转录因子,因此c-Abl对FoxM1的调控机制值得探讨。我们前期的研究发现,过表达的c-Abl能够结合并且磷酸化FoxM1,我们通过质谱分析(LC-MS/MS)鉴定出5个主要的磷酸化位点(分别为Y129、Y272、Y317、Y362、Y575)。本研究将对两者的相互作用,c-Abl对FoxM1的磷酸化修饰进行更深入的探讨,并着重研究c-Abl调控FoxM1的分子机制和生理意义,包括泛素化调控、表达水平调控和转录功能调控,以及这些调控作用对细胞周期进程和细胞凋亡的影响。在本研究中,我们首先使用免疫共沉淀、免疫印迹和激光共聚焦等技术手段,证实内源性的c-Abl与FoxM1存在相互作用,相互作用主要定位于细胞核,且主要发生于有丝分裂期(M期)。进一步,我们证实内源性FoxM1可被酪氨酸磷酸化修饰,并且c-Abl激酶特异性抑制剂STI571能够显著抑制FoxM1酪氨酸磷酸化。接下来,我们研究c-Abl对FoxM1表达水平的影响,发现c-abl/arg被敲低或敲除后,FoxM1的表达水平也出现明显下调;同时发现,使用STI571抑制细胞中的c-Abl激酶活性,也能够显著下调FoxM1的表达水平,说明FoxM1的表达依赖于c-Abl激酶活性。我们使用放线菌酮(CHX)处理细胞,抑制蛋白质合成,检测MEF WT细胞和MEF(c-abl-/-/arg-/-)细胞中FoxM1蛋白的半衰期,发现c-abl/arg敲除后,FoxM1的半衰期显著缩短,说明c-Abl/Arg能够增强FoxM1的蛋白稳定性。我们猜测,c-Abl对FoxM1的磷酸化调控其经泛素--蛋白酶体途径降解,导致其表达水平受到影响。为此,通过使用STI571和蛋白酶体抑制剂MG132处理U2OS细胞,检测FoxM1的泛素化程度和蛋白表达水平,发现STI571处理显著增强其泛素化程度并下调其表达水平;而MG132处理能够使其表达水平明显回升,说明c-Abl能够抑制FoxM1的泛素-蛋白酶体降解途径,从而提高FoxM1的表达水平。进一步,我们对FoxM1磷酸化突变体的泛素化修饰和表达水平进行鉴定,发现与野生型FoxM1相比较,Y272F、Y575F及Y272/575F突变体的泛素化程度显著增强,表达水平明显下调,同时与E3泛素连接酶组件CDH1的结合作用显著增强,说明c-Abl对Y272和Y575这两个位点的磷酸化,对FoxM1的稳定性有重要作用。同时发现Y317F、Y362F突变体的表达水平明显升高,并且CDH1与它们的结合作用显著减弱,提示c-Abl对Y317、Y362的磷酸化可能促进FoxM1蛋白的降解。FoxM1通过调节大量的关键基因参与细胞周期调控,这些基因蛋白包括Cyclin B1、Cyclin D1、CDC25A、Plk1、CENP-F、Aurora-A及Aurora-B等。既然c-Abl能够磷酸化并调控FoxM1表达水平,那么对FoxM1转录功能的影响也值得探讨。我们发现,当abl/arg被敲低后,细胞中的FoxM1及其下游靶基因蛋白Cyclin B1、Cyclin G2、Plk1的表达水平显著下调。同时,使用STI571抑制c-Abl的激酶活性,也能够显著下调Cyclin B1、Plk1和CENP-F等的转录水平和表达水平,说明c-Abl对FoxM1的调控能够显著影响其转录功能。进一步,我们探讨c-Abl对FoxM1的调控能否影响细胞周期进程。通过使用细胞周期同步化和流式细胞仪检测等技术方法,我们证实STI 571通过抑制c-Abl激酶活性,下调FoxM1的表达水平,并抑制其转录功能,进而抑制其靶基因蛋白Cyclin B1、Plk1和Aurora-B等的转录表达,最终导致细胞周期阻滞。FoxM1的另一个重要功能是参与肿瘤细胞的抗凋亡,为此,我们研究c-Abl对FoxM1的调控是否影响肿瘤细胞的凋亡。通过使用STI571和阿霉素(Doxorubicin,能够诱导肿瘤细胞凋亡)处理Hela细胞,检测凋亡蛋白Cleaved caspase3和Cleaved PARP-1的表达水平,发现STI571处理加剧了阿霉素诱导的Hela细胞凋亡,说明STI571通过抑制c-Abl激酶活性,下调FoxM1表达水平,抑制FoxM1的抗凋亡功能。我们的研究证实,c-Abl能够明显调控转录因子FoxM1,并且根据文献报道,c-Abl也能够调控其他的转录因子如c-Myc、NF-κB、C/EBPβ等。因此我们猜测,c-Abl/Arg能够参与调控基因的转录表达。为此,我们使用高通量测序(RNA-seq)和生物信息学分析的方法,对MCF-7WT及MCF-7 KD(c-abl/arg Knock-Down)细胞的RNA进行转录组分析。发现共有1034个基因的转录水平受到c-Abl/Arg的显著调控,c-abl/arg敲低之后,有635个基因的转录水平下调,399个基因的转录水平上调,说明c-Abl/Arg能够调控基因的转录水平,并且以正向调控为主。进一步,我们对受c-Abl/Arg调控的基因进行功能聚类分析,发现它们的功能参与多种信号通路和细胞代谢进程,并且这些通路和进程大多与已报道的c-Abl功能密切相关,提示c-Abl不仅在蛋白水平对相关功能通路进行调控,并且在基因转录水平也参与调控。综合以上研究结果,我们证实细胞内源性的c-Abl能够结合并磷酸化修饰FoxM1;c-Abl对FoxM1的磷酸化作用,抑制其经泛素-蛋白酶体途径降解,增强其稳定性,上调其表达水平;c-Abl对FoxM1的调控影响细胞周期进程和肿瘤细胞凋亡;c-Abl/Arg能够参与基因转录水平调控。本研究为深入探讨c-Abl和FoxM1的功能,特别是探讨两者在细胞周期进程和肿瘤细胞凋亡等方面的功能,提供重要的理论支撑。
[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.

【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2017
【分类号】：Q78

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