导　　师： 关洪斌;董文吉

学科专业： 071010

授予学位： 硕士

作　　者： ;

机构地区： 山东大学

摘　　要： Akt是一种丝氨酸／苏氨酸蛋白激酶,作为P13K／Akt信号通路的关键分子,能够通过磷酸化激活或抑制其下游一系列底物如GSK-3、Bad caspase9 NF-κB和p27kipl等参与细胞增殖、分化、凋亡和代谢的调节。Akt在肿瘤发生及转移中也起着重要作用,它的持续活化能够抑制肿瘤细胞的凋亡,促进肿瘤的生长。因此深入研究Akt调节机体正常的生理状态和参与疾病发生的分子机制也成为当前的热点。 核基质结合蛋白SATB1 /(special AT-rich sequences binding protein 1, SATB1/)能够自身多聚化,围绕异染色质形成笼状结构分布在细胞核中,SATB1不仅结合染色质DNA的核基质结合区/(matrix attachment regions, MARs/),也结合核基质/(nuclear matrix/),能够使DNA锚定在核基质并形成环状结构/(loop/)。SATB1的磷酸化、乙酰化和小泛素化样修饰/(sumoylation/)可调节其DNA结合能力和细胞核内亚结构的定位；SATB1与多种蛋白质相互作用,能够募集染色质重塑复合物和组蛋白修饰酶,实现对其靶基因表达的时空特异性调控。SATB1在调节细胞分化、细胞凋亡、肿瘤生长与转移和X染色体失活等方面起着重要作用。 已有研究证实SATB1能被PKC磷酸化,SATB1的这种磷酸化状态与其转录调节能力相关。根据现有的研究和Scansite的数据我们推测SATB1能够被Akt磷酸化。由数据显示SATB1含有2个候选磷酸化位点,分别是S47/(RGRLGST/)和S557/(IRRFLSL/)。首先构建重组质粒pcDNA4//SATB1 wt,利用免疫沉淀方法及免疫印迹法验证蛋白激酶Akt1与野生型SATB1分子间的激酶底物关系；接下来将SATB1分子的47位点的丝氨酸进行定点突变,获得pGEX4T-1//SATB1 S47A、pGEX4T-1//SATB1 S47D,应用体外激酶活性分析方法确定SATB1分子被Akt1磷酸化的位点。同时还设计了免疫共沉淀方法和GST-pull down实验鉴定Akt1与SATB1分子间是否存在相互作用。最后我们构建了pGFP-N2//SATB1,去探索SATB1� Akt is a serine//thronine kinase. The protein kinase Akt acts as the major downstream target of P1-3K and regulates the biological function of cells. Those Akt substrates,such as GSK-3、Bad、caspase9、NF-κB and p27kip1, that are most likely to contribute to the diverse cellular roles of Akt, which include cell survival, growth and proliferation. Indeed, recent studies have revealed that Akt plays important role in tumorigenesis and metastasis. Frequent aberrant activation of Akt can promote tumorigenesis by blocking apoptosis. The important role of Akt in human physiology and disease has made it a researching target. SATB1 /(special AT-rich sequences binding protein 1/) is polymerized and forms a cage-like structure surrounding heterochromatin, whereby it tethers nuclear matrix and matrix attachment region through direct binding, dynamically orchestrating chromatin DNA loop formation. Signaling events trigger phosphorylation, acetylation, and sumoylation on SATB1, thereby either modulating its DNA binding ability or changing its intranuclear localization. SATB1 associates with a number of molecules, recruits chromatin remodeling complexes and histone modifying enzymes and regulates gene expression temporospatially. This review highlights the roles of SATB1 in cell differentiation, apoptosis, tumor growth, metastasis, and X chromosome inactivation. SATB1 holds promise in clinical therapeutics, particularly cancer metastasis treatment. Previous studies have confirmed that SATB1 can be phosphorylatied by PKC which is related to SATB1 transcription ability. We speculate that the human and mouse SATB1 can be phosphorylated by Akt1 based on existing research and data of Scansite. As the data shown, SATB1 contains three candidate phosphorylation sites, namely S47 /(RGRLGST/) and S557 /(IRRFLSL/). Our research aims at exploring the relationship between Akt1 and SATB1. First, the cDNA encoding human SATB1 was sub-cloned into expression vector pcDNA4 We explored the relationship between Aktl and SATB1 using

关 键 词： 磷酸化 免疫沉淀 免疫印迹

领　　域： [生物学]