蛋白激酶CK2与转录因子Pdx-1的相互作用研究

发布时间：2018-03-31 23:00

本文选题：磷酸化　切入点：蛋白质相互作用　出处：《华中科技大学》2010年博士论文

【摘要】： 蛋白质的磷酸化修饰是生物体内最重要翻译后修饰(post-translational modification)方式之一,在哺乳动物细胞中,至少有1／3的蛋白质发生过磷酸化,因而其作用几乎涵盖了所有的生理及病理过程,尤其在细胞的信号传递过程中占有及其重要的地位。蛋白质的磷酸化过程是在蛋白激酶的催化下,由ATP或GTP提供磷酸基及能量而完成的。人类基因组约2%的基因编码了518种蛋白激酶5,作为最早被鉴定出的且最为重要的蛋白激酶之一,酪蛋白激酶2(casein kinase 2, CK2)是一种高度保守的、在各种真核生物中都广泛存在,并且具有多种生理功能的丝氨酸／苏氨酸激酶。CK2是由两个催化亚基(α和α')和两个调节亚基(β)组成的异四聚体(α2β2,α'2β2或αα'β2)；其广泛的参与了细胞正常功能的调控,如基因表达,蛋白合成,细胞周期调控等,同时还参与如癌症发生,抗凋亡等病理过程。2003年已报道CK2有多达307种作用底物,其中约五分之一的底物为调节基因表达的转录因子。胰十二指肠同源盒-1(Pancreatic duodenal homeobox-1, Pdx-1)是由Hox样同源盒基因编码的转录因子。Pdx-1是参与包括胰岛素基因在内的多个β细胞特异性基因转录的主控转录因子。生理方面,Pdx-1参与了胰腺的发育和分化,促进胰岛β细胞增殖、成熟和功能的维持；病理方面,除了Pdx-1的基因变异与糖尿病的发生、发展密切相关外,还发现Pdx-1在包括胰腺癌、结肠癌、肺癌和乳腺癌在内的多种肿瘤细胞中呈现过表达,且认为Pdx-1在肿瘤细胞中有促增殖效应,可能与肿瘤的发生、发展、转移及对化疗的耐药有关。鉴于Pdx-1的功能如此关键,阐明其在细胞内表达和激活的分子调控机制已成为多个研究领域内的一个热点问题。近几年越来越多的研究表明,Pdx-1为细胞内可磷酸化蛋白,且翻译后磷酸化修饰是调节Pdx-1活性的一个重要方式。本研究通过体内、外磷酸化实验证明,CK2激酶可磷酸化Pdx-1蛋白以及通过体外定点突变技术寻找到了Pdx-1蛋白的磷酸化位点即位于其羧基末端的thr231和ser232。并进一步通过荧光素酶活性分析实验及蛋白合成抑制剂放线菌酮(CHX)和蛋白酶体抑制剂MG132实验发现,CK2激酶磷酸化Pdx-1后不仅可抑制其对胰岛素基因的转录活性；而且可影响其自身蛋白的稳定性,使其更易于受蛋白酶体依赖性(proteasome-dependent)蛋白降解途径的降解；且该作用可能是导致抑制其转录活性的原因之一。另一方面,采用免疫荧光实验,通过激光共聚焦显微镜我们发现,虽然CK2激酶的磷酸化作用并未影响Pdx-1蛋白的亚细胞定位；但是Pdx-1与CK2激酶各亚基(α,α',β)荧光图片在细胞核内的重叠表明他们在核内共定位,因而提示Pdx-1与CK2激酶可能存在分子间相互作用。体外利用GST Pull-down、Far-western实验和在体内用免疫共沉淀实验分别进一步证明了Pdx-1蛋白可与CK2激酶各亚基和全酶发生特异性结合。综上所述,本研究从分子、细胞水平上提供了CK2激酶和Pdx-1蛋白磷酸化和相互作用的实验依据,并进一步探讨了该作用的重要生物学意义,有助于我们理解Pdx-1在参与细胞事件中的空间和时间调控机制。
[Abstract]:Protein phosphorylation is the most important in vivo post-translational modification (post-translational modification) and one of the ways in mammalian cells, at least 1 / 3 protein phosphorylation occurred, because it covers almost all of the physiological and pathological processes, in particular, and play an important role in the process of cell signaling. Phosphorylation of protein in catalytic protein kinase, by ATP or GTP with phosphate and energy and the completion of the human genome. Gene encoding about 2% of the 518 protein kinase 5, identified as the first and one of the most important protein kinase, casein kinase 2 (casein kinase 2. CK2) is a highly conserved and widely exists in various eukaryotic organisms, and a variety of physiological functions of serine / threonine kinase.CK2 is composed of two catalytic subunits (alpha and alpha ') and two The regulatory subunit (beta) ISO four dimer composition (alpha 2 beta 2, alpha'2 beta 2 and alpha beta alpha '2); the regulation of extensively involved in the normal function of cells, such as gene expression, protein synthesis, cell cycle regulation, is also involved in such as cancer pathology, anti apoptosis etc. the process of.2003 CK2 has been reported to have as many as 307 kinds of substrates, of which about 1/5 of the substrate as a transcription factor to regulate gene expression.
Pancreatic duodenal homeobox -1 (Pancreatic duodenal homeobox-1, Pdx-1) is a transcription factor.Pdx-1 by Hox like homeobox gene encoding the main transcription factors involved in multiple beta cell specific transcription of genes including the insulin gene. Physiological aspects, Pdx-1 is involved in pancreas development and differentiation, promote beta cell proliferation. Maintain the maturation and function of Pathology; besides, Pdx-1 gene mutation and the occurrence of diabetes, is closely related to the development, also found in Pdx-1 including pancreatic cancer, colon cancer, lung cancer and breast cancer, tumor cells overexpress, and that Pdx-1 has the proliferation promoting effect in tumor cells, and tumor the occurrence, development, metastasis and resistance to chemotherapy. In view of the function of Pdx-1 is so critical to elucidate its expression in cells and activation of the molecular regulation mechanism has become an area of research A hot issue.
In recent years, more and more research shows that Pdx-1 cells can be phosphorylated proteins and post-translational phosphorylation is an important way to regulate the activity of Pdx-1. This study through the body, outside the phosphorylation experiments show that CK2 can be phosphorylated Pdx-1 protein kinase and by in vitro mutagenesis to find phosphorylation sites Pdx-1 protein is located at the C-terminal of thr231 and ser232. and further by luciferase reporter assay and the protein synthesis inhibitor cycloheximide (CHX) and proteasome inhibitor MG132 experiment, CK2 kinase phosphorylation after Pdx-1 can not only inhibit the insulin gene transcription activity; stability but also can influence its own protein. To make it more susceptible to proteasome dependent degradation pathway of protein degradation (proteasome-dependent); and the effect may be the cause of inhibition of its transcriptional activity On the other hand, by immunofluorescence experiments by confocal laser scanning microscopy we found that although CK2 kinase phosphorylation did not affect the subcellular localization of Pdx-1 protein; but Pdx-1 and CK2 kinase subunits (alpha, alpha ', beta) fluorescence images overlap table in the nucleus that they co localization in the nucleus therefore, suggested that Pdx-1 CK2 kinase and possible molecular interactions in vitro. Using GST Pull-down, Far-western assay and in vivo immune experiments were further proved that Pdx-1 protein could bind with CK2 kinase subunits and the enzyme specificity of co precipitation. In summary, this study from the molecular, cellular level provides the experimental basis for CK2 kinase and Pdx-1 protein phosphorylation and interaction, and further discusses the important role of the biological significance, contribute to our understanding of the involvement of Pdx-1 in cellular events in space and time control Mechanism.

【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R363

【参考文献】