【作者】 刘宁；

【导师】 王平；

【作者基本信息】 华东师范大学 ， 生物医学， 2011， 博士

【摘要】 KLFs家族是一类高度保守的、含锌指结构的蛋白质,通过调节富含GC的启动子的表达,在细胞增殖、细胞凋亡、细胞分化、血管生成、淋巴管生成、个体发育、肿瘤生成及胚胎干细胞发育等过程中发挥着重要作用。目前已经发现17个家族成员,每个成员在细胞中的生物学功能并不相同。越来越多的报道指出KLFs蛋白的表达异常与多种肿瘤的发生相关,但是具体的分子机制仍有待于进一步的研究。其中KLF2、KLF、KLF5在生理及病理过程中如细胞增殖、干细胞自我更新与分化、脂肪细胞的分化及肿瘤的发生等过程中发挥着重要作用,近年来,KLF2、KLF4、KLF5已经成为了KLFs家族中的研究热点。已有文献报道KLF5在脂肪细胞分化、干细胞分化及自我更新中有十分重要的功能,而且能够促进许多肿瘤细胞如结肠癌、乳腺癌细胞的增殖并抑制细胞凋亡,被认为是一种致癌基因；KLF2在T淋巴细胞的迁移过程中起着重要的作用,还能够抑制脂肪细胞的分化,调节胚胎干细胞的自我更新；缺失KLF2、KLF4及KLF5,胚胎干细胞的全能性及自我更新的能力会丧失。因此研究它们在细胞内的稳定性对于维持机体正常的生物学功能有着重要的意义。然而到目前为止,关于KLFs的报道大多集中在其转录水平的调控研究,对其蛋白质水平的修饰及分子机制的研究报道较少。因此,研究KLFs家族蛋白稳定性及相关生物学功能对于理解肿瘤发生过程,胚胎个体发育及相关疾病的治疗都有着重要的意义。SCFFbw7是近年来报道的在肿瘤发生、细胞增殖及分化过程中起重要作用的泛素连接酶。SCFFbw7是由Fbw7、SKP1及CUL1蛋白组成的复合体,能够与底物特异的结合并通过泛素化依赖的蛋白酶体途径介导靶蛋白的降解。其中,Fbw7(又名FBXW7, CDC4, Sel10, Ago)是一个从酵母到哺乳动物中都高度保守的F-box蛋白,能够通过识别底物上保守的CPD降解信号肽：S/TPXXT/D/E来降解靶蛋白。越来越多的证据表明Fbw7是一个重要的肿瘤抑制因子,在细胞增殖、分化及生长过程中起着非常重要的作用。Fbw7能够降解许多与细胞生长及增殖密切相关的蛋白,比如c-Myc、Cyclin E、Notch及c-Jun等。在许多肿瘤如乳腺癌、肠癌、胃癌、肺癌等,Fbw7发生突变或缺失,导致一些癌蛋白如c-Myc, c-Jun等在细胞内聚集而使细胞生长失控,最终促使肿瘤的形成。尽管近年来Fbw7作为肿瘤抑制因子的生物功能得到了广泛的研究,但其抑制肿瘤发生的具体分子机制及其新的生物学功能仍然有待于进一步研究。在本论文中,我们运用生物信息学、细胞生物学、分子生物学的技术筛选鉴定了调控KLF5、KLF2蛋白降解的一种新的E3连接酶即肿瘤抑制因子Fbw7。本课题重点研究了KLF5蛋白稳定性,从生化、细胞、分子水平上研究了Fbw7调控KLF5蛋白降解的具体分子机制及新的生物学功能；同时也初步研究了Fbw7调控KLF2蛋白降解的分子机制。本论文主要分成以下几部分：一、Fbw7调控KLF5及KLF2蛋白的稳定性研究1.我们通过生物信息学的方法,运用Scansite软件对KLFs蛋白进行序列扫描,发现转录因子KLF2, KLF5含有典型的Fbw7降解信号肽CPD即S/TPXXT/D/E。提示我们,它们有可能被Fbw7降解。2.研究外源的Fbw7对KLF5及KLF2蛋白水平的调控。我们在293T细胞中分别共表达KLF2、KLF4、KLF5及Fbw7、β-TrCP1、Fbw2、Fbw5、Fbw8等一系列E3泛素连接酶,利用Western Blot技术证明只有Fbw7能够明显抑制KLF2及KLF5蛋白水平的表达,对KLF4蛋白水平的影响较小。这种降解是剂量依赖的,且是能够被蛋白酶体抑制剂MG132所抑制的。而且我们还利用流式细胞仪的技术进一步验证了Fbw7确实能够降解KLF2、KLF5蛋白水平的表达,而且这种降解同样能够被MG132所抑制,提示Fbw7降解KLF2、KLF5可能是通过蛋白酶体途径降解。3.研究内源的Fbw7对KLF5蛋白水平的调控。过表达Fbw7后,利用免疫荧光技术检测Hela细胞内源KLF5蛋白水平的变化。用siFbw7抑制Hela细胞内源Fbw7表达后,再用RT-PCR, Western Blot检测mRNA和蛋白水平上KLF5表达的变化,发现KLF5的mRNA水平没有什么变化,但是蛋白水平表达明显升高。利用Pulse chase同位素标记实验检测内源KLF5的半衰期,发现抑制内源Fbw7表达后,KLF5的半衰期明显延长。利用免疫荧光、Western Blot检测HCT116 Fbw7-/-, DLD1Fbw7-/-两种缺失细胞及HCT116, DLD1野生型细胞中的KLF5蛋白水平,发现缺失Fbw7的细胞中,KLF5蛋白明显高表达。上述实验结果初步证明KLF2及KLF5确实是Fbw7的新的降解底物,不论是外源的Fbw7还是内源的Fbw7都能够降解KLF5及KLF2。下一步我们就对Fbw7调控KLF2及KLF5的具体生化分子机制进行了一系列的研究。二、Fbw7降解KLF5及KLF2的生化分子机制研究1.利用免疫共沉淀技术证明Fbw7与KLF2、KLF5的体内相互作用,Pull Down的方法检测体外直接相互作用。2.确定KLF5、KLF2与Fbw7相互作用的具体作用位点。首先我们构建了KLF5、KLF2及Fbw7各种突变体,在293T细胞中共表达后,利用Western Blot、免疫共沉淀技术鉴定相互作用结构域,发现了KLF5的227位苏氨酸、292位丝氨酸、312位苏氨酸,KLF2的243位苏氨酸、247位丝氨酸是Fbw7的WD40结构域的潜在作用位点。3.研究Fbw7调控KLF5、KLF2降解的具体分子机制。利用体内泛素化实验证明了Fbw7确实能够促进KLF5、KLF2的泛素化。在Hela细胞内抑制Fbw7表达,内源KLF5蛋白的泛素化明显减弱。上述实验结果证明Fbw7能够通过泛素-蛋白酶体途径来促进KLF5及KLF2的泛素化降解。三、KLF5磷酸化与Fbw7介导的蛋白降解1.利用免疫共沉淀、体外磷酸化实验证明KLF5能够与GSK3β蛋白激酶在体内相互作用,被GSK3β磷酸化。2. Western Blot实验证明过表达GSK3β能够促进KLF5蛋白的降解。Pulse chase实验证明干扰RNA抑制GSK3β表达后,能够延长KLF5的半衰期。3.流式细胞仪、免疫共沉淀、体内泛素化的实验证明用GSK3特异性抑制剂LiCl抑制其激酶活性后,能够抑制Fbw7与KLF5的相互作用及其对KLF5的泛素化降解。上述结果证明磷酸激酶GSK3β能够通过磷酸化KLF5的CPD来促进Fbw7介导的KLF5降解。四、Fbw7介导的KLF5蛋白降解及其生理功能的研究1.研究Fbw7降解KLF5与肿瘤的关系。KLF5在肿瘤细胞中高表达,而且过表达KLF5会促进细胞增殖。为了确定Fbw7是否会抑制KLF5的生物功能,我们将KLF5与Fbw7或Fbw7-F-box的缺失突变体共表达在结肠癌细胞HCT116中,利用肿瘤细胞集落形成实验检测Fbw7对KLF5引起的细胞增殖的影响,结果表明Fbw7能够抑制KLF5引起的细胞增殖。此外我们还用siRNA抑制内源的KLF5,然后,用瞬时转染或逆转录病毒的方法将野生型或CPD突变的KLF5表达入HCT116中,结果发现过表达KLF5野生型的细胞与对照组的细胞相比,细胞集落形成较多,而过表达CPD突变的KLF5的细胞集落形成最多。共表达Fbw7后,能够抑制KLF5诱导的细胞增殖,但是对CPD突变的KLF5诱导的细胞增殖抑制作用很小。MTT的实验结果同样验证了Fbw7介导的KLF5降解能够抑制KLF5引起的肿瘤细胞的增殖。2.研究Fbw7降解KLF5对KLF5转录水平调控下游靶基因的影响。我们用RT-PCR的方法检测KLF5调控的下游基因在Fbw7野生型及缺失的细胞中的表达,结果表明在缺失了Fbw7的细胞中Survivin的mRNA水平明显高表达,说明Fbw7能够通过调控KLF5而调控下游基因。此外我们还用荧光素酶报告基因的方法检测发现KLF5能够增强Survivin基因启动子的活性,然而共转了Fbw7后,Survivin基因启动子的活性被抑制。上述实验结果证明了Fbw7能够抑制KLF5下游抗凋亡基因Survivin启动子的活性,抑制KLF5引起的肿瘤细胞的增殖。综上所述,我们运用生物信息学、生化及分子生物学技术、细胞生物学技术成功地筛选并鉴定了调控KLF5、KLF2蛋白降解的一种新的泛素连接酶即肿瘤抑制因子Fbw7,首次发现了Fbw7抑癌蛋白对KLF5以及KLF2蛋白水平的负调控机制。对于进一步理解KLF5及KLF2的蛋白稳定性及生物功能提供了新的线索和思路。本课题着重研究了Fbw7对KLF5蛋白稳定性的调控,发现肿瘤抑制因子Fbw7能够通过磷酸化依赖的途径,促进促细胞生长因子KLF5的泛素化降解,在转录水平上抑制KLF5调控的下游基因的表达,细胞水平上抑制了结肠癌细胞的增殖。KLF5在肿瘤发生当中扮演着重要的角色,发现Fbw7对KLF5蛋白水平的负调控,对于理解肿瘤的发生具有重要意义,可以为治疗肿瘤及其他疾病提供新的靶点。此外,本课题也为下一阶段筛选Fbw7新底物及其相关生物学功能的研究提供了有利的技术平台。更多还原

【Abstract】 Kruppel-like factors (KLFs), the zinc finger containing protein family, play an important role in cell proliferation, apoptosis, cell differentiation, angiogenesis, lymphogenesis, tumorigenesis, as well as in embryonic stem cell (ES cell) development through the regulation of various rich GCs gene promoters Each member of the KLFs family has its unique biological functions and more than 17 family members have been identified. A lot of evidence showed that the abnormal expression of KLFs is correlated with tumorigenesis. However, the detailed mechanisms remain unknown. Recently, KLF2, KLF4, and KLF5 have become the research focus within the KLFs family, because of their functions in cell differentiation, self-renewal, adipocyte differentiation, and tumorigenesis. KLF5 was related to adipocyte, stem cells differentiation and self-renewal. Moreover, it has been regarded as an oncoprotein which is able to promote tumor cell proliferation. KLF2, another member of KLFs, is involved in T cell egress, inhibits adipocyte differentiation, and regulates the self-renew and proliferation of ES cells. KLF2,4, and 5 deficient ES cells lose their totipotency and ability of self-renew. Yet, most studies focus on the transcriptional regulation of the KLFs. Their post-transcriptional modulation remains to be largely unknown.SCFFbw7, an E3 ubiquitin ligase, has a critical role in tumorigenesis, cell proliferation and cell differentiation. This protein complex consists of Fbw7, SKP1, and CUL1 and mediates the degradation of its target protein via ubiquitination dependent pathway. Fbw7, also called FBXW7, CDC4, Sel10, Ago, one of the F-box proteins plays a critical role in identifying its substrates. Fbw7 conserves from yeast to mammalian. Fbw7 degrades its substraes through a conserved concensus CPD(CDC4 phospho-degron):S/TPXXT/D/E. More evidence suggest that Fbw7, an important tumor suppressor, plays a vital role in cell proliferation, differentiation and cell growth. For example, Fbw7 is able to degrade c-Myc, Cyclin E, Notch and c-Jun and these targets are highly related to cell proliferation. Furthermore, clinical samples from bowel, lung and breast cancer, also contain Fbw7 mutations and accumulation of c-Myc and c-Jun. However, the specific molecular mechanisms through which Fbw7 functions as a tumor inhibitor need to be unveiled in future research. Study of mechanism will provide a novel theoretical idea for tumor treatment.In this article, we identified tumor suppressor Fbw7 as a novel E3 ligase for KLF5 and KLF2 using bioinformatics, cytobiology and molecular biology techniques. We further studied the mechanism by which Fbw7 mediates the degradation of KLF5.The major parts of our research are as following:1. Fbw7 regulates the stabilities of KLF5 and KLF2 1) Through the analysis of KLFs family proteins using Scansite software, we idenfied the CPD:S/TPXXT/D/E, a classical signal for Fbw7 dependent degradation in KLF2 and 5.2) To study the degradation of KLF5 by exogenous Fbw7, we overexpressed KLF2, KLF4, KLF5 and Fbw7α, p-TrCP1, Fbw2, Fbw5, Fbw8 in HEK293T cell. Our data showed that only Fbw7a degraded KLF2 and 5, and MG132 treatment inhibited the degradation. This result was confirmed by flow cytometre. Together, our data indicated that Fbw7αdegrade KLF2 and 5 via proteasome degradation pathway.3) To study the degradation of KLF5 by endogenous Fbw7, we transfected siFbw7 into Hela cells to knockdown endogenous Fbw7. Our data showed knocking down Fbw7 up-regulated of KLF5 protein levels, while had minor effect on KLF5 mRNA levels. Moreover, knockdown of Fbw7 delayed the turnover of endogenous KLF5 via the pulse chase assay. The protein levels of KLF5 in Fbw7-deficient HCT116 or DLD1 cells are much higher than their parent cells.Taken together, our data clearly showed that both endogenous and exogenous Fbw7 can efficiently degrade KLF5 and 2.2. The molecular mechanisms of the degradation of KLF2 and 5 by Fbw7.Next, we focused on the molecular mechanisms by which Fbw7 mediated KLF2 and 5 degradation.1) Immunoprecipitation and Pull Down assays were used to detect the interaction between Fbw7 and KLF2 and 5 in vivo and in vitro.2) To identify the domains of interaction between KLF2,5 and FBW7, different mutations were transfected into HEK293T and our data indicated that the T227, S292, T312 sites of KLF5 and the T243, S247 sites of KLF2 are the potential sites for interaction.3) We tested whether Fbw7 promotes KLF2 and KLF5 ubiquitination using an in vivo and in vitro ubiquitination assay. The results showed that Fbw7 can promote the ubiquitination of KLF2 and 5. Knocking down of Fbw7 inhibited the KLF5 ubiquitination.All the above showed that Fbw7 regulated the degradation of KLF2 and 5 via Ubiquitin-proteasome pathway.3. GSK3p is required for the Fbw7-mediated degradation of KLF5.1) KLF5 is able to be phosphorylated by GSK3P in vitro phosphorylation experiment.2) Overexpression of GSK3βcould promote the degradation of KLF5 and knockdown of GSK3βdelayed the turnover of endogenous KLF5 via the pulse chase experiment.3) To further understand the mechanism by which GSK3βaffects KLF5 stability, we examined the effect of GSK3βactivity on the interaction between Fbw7 and KLF5. Treatment with LiCl clearly decreased the interaction between endogenous Fbw7 and KLF5 and also significantly inhibited the ubiquitination of endogenous KLF5Our data showed that GSK3βcan promote the degradation of KLF5. 4. Fbw7-mediated degradation regulates KLF5 biological activity1) Fbw7 inhibits KLF5-promoted cell proliferation. Overexpression of KLF5 promoted cell proliferation. We tested whether Fbw7-mediated degradation inhibited KLF5-dependent cell proliferation using a Soft Agar formation assay. KLF5, Fbw7 or its CPD mutation was transfected into HCT-116 cells. Compared to empty vector, more colonies were formed when wild-type KLF5 was overexpressed, whereas the Fbw7-resistant mutant KLF5-3A was able to stimulate HCT116 cells to form colonies more than the wild type. In addition, co-expression of Fbw7significantly inhibited KLF5-mediated cell proliferation, but had little effect on KLF5-3A-mediated proliferation. Furthermore, The results of MTT also showed that expression of mouse KLF5-3A had a stronger effect on cell proliferation than did the wild type.2) Fbw7 inhibits KLF5 transcriptional activity. Based on our results thus far, we speculated that Fbw7 might inhibit the expression of KLF5-transactivated genes. We therefore measured by RT-PCR the expression levels of the known KLF5-targeted genes in wild-type or Fbw7-deficient HCT116 and DLD1 cancer cells. Survivin mRNA levels in particular were elevated significantly in both types of Fbw7-deficient cells. Subsequent luciferase reporter assays showed that the expression of KLF5 significantly enhanced the activity of the survivin essential promoter in HCT116 cells, whereas this effect was inhibited efficiently by co-expression of Fbw7.Thus, Fbw7 appears to downregulate the transcriptional activity of KLF5 and inhibits KLF5-promoted the colorectal cancer cells proliferation.Taken together, we have identified the novel substrates KLF5 and KLF2 of E3 ligase, Fbw7, via the tools of bioinformatics, cytobiology and molecular biology. For the first time we revealed that the tumor suppressor Fbw7 can down-regulate KLF5 and KLF2 on their protein level. Our work contributes some new clues and methods for the understanding of KLF5 and KLF2 protein stability and biological function. This research identified a novel substrate KLF5 of E3 ligase Fbw7 and the molecular mechanism of the degradation of KLF5 by Fbw7. In conclusion, Fbw7 could promote the degradation of KLF5 via Ubiquitin-proteasome, inhibit the expression of KLF5 downstream genes and the colorectal cancer cells proliferation. KLF5 plays a very important role in tumorigenesis. This new regulatory mechanism of KLF5 degradation may provide significant plateform, where we can learn useful diagnostic and therapeutic targets for tumor and efficacious methods for identifying the new substrates of Fbw7.更多还原