【作者】 孟睿；

【导师】 伍钢；

【作者基本信息】 华中科技大学 ， 肿瘤学， 2010， 博士

【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。更多还原

【Abstract】 Phosphorylation is one of the most important mechanisms for the post-translational modification of proteins. Studies of mammalian cells metabolically labeled with [32P] orthophosphate suggest that as many as one-third of all cellular proteins are covalently modified by protein phosphorylation.Phosphorylation can regulate almost every property of a protein and is involved in all fundamental cellular processes, of particular importance is its involvement in signal transduction. The process of phosphorylation is under the control of protein kinases by transfering a phosphate group from ATP/GTP to the specific substrate protein.The human genome contains 518 protein kinase genes and they constitute about 2% of all human genes. As one of the first identified protein kinases, Casein kinase 2 is a highly conserved, ubiquitously expressed, serine/threonine protein kinase that is a heterotetramer (α2β2,α’2β2orαα’β2)composed of two catalytic subunits(αorα’)and two regulatory subunits(β).CK2 has been implicated in various physiological and pathological cellular processes, such as gene expression, protein sythesis, cell cycle regulation as well as anti-apopotosis, carcinogenesis and etc. Until the year 2003, there are up to 307 proteins identified as CK2 substrates and more than one-fifth of these are implicated in gene expression as transcriptional factors.Pdx-1 (Pancreatic and duodenal homeobox-1) is a homeodomain transcription factor and functions as a masterfactor in the transcription of a large amount ofβ-cell specific genes including the insulin gene. Physiologically, Pdx-1 is necessary for pancreatic development and differentiation. In addition, it is also involved in the maintainance of adultβ-cell function. Pathologically, Pdx-1 gene mutations were closely related to the onset and development of diabetes.Furthermore, recent findings also revealed that Pdx-1 was overexpressed in various cancers including pancreatic cancer, colorectal cancer, lung cancer, breast cancer and etc.It was stated also in these findings that Pdx-1 was related to the survival of some of these cancer cells and thus supposed to be implicated in the carcinogenesis, development, metastasis and drug resistanse of cancers. It is thus crucial and a hot topic to determine the molecular mechanisms involved in the regulation of Pdx-1 expression and activation.There is increasing evidence showing that Pdx-1 is a phosphoprotein and that post-translational phosphorylation plays important roles in the regulation of some of its functions.In the present study we identified Pdx-1 as a bona fide substrate for CK2 by both in vitro phosphorylation assays and in vivo labelling of cells with phosphate, additional site-directed mutation analysis of Pdx-1 enabled us to find out the specific phosphorylation sites for CK2, namely Ser232 and Thr231,on the C-terminal region of Pdx-1 protein.Most interestingly, by employing luciferase reporter assay, cycloheximide(protein sythesis inhibitor) treatment and MG132(proteasome inhibitor) treatment experiments, we demonstrated that the phosphorylation of Pdx-1 by CK2 decreased its transcriptional activity on the insulin promoter, and on the other hand, CK2 phosphorylation also drived Pdx-1 to degradation which is probably in a proteasome dependent manner.Further immnofluroscence studies revealed that CK2 phosphorylation was not implicated in the regulation of the subcellular localization of Pdx-1. In contrast, overlay images showed a colocalization of Pdx-1 and individual CK2 subunits (α,α’,β) in the nucleous, suggesting that there might be inter-molecular interactions between these proteins. Therefore, GST-pull down and Far-western blot assays as well as immunoprecipitation experiments were performed to identify the protein protein binding between Pdx-1 and CK2. We demonstrated that Pdx-1 directly interacted with both the CK2 holoenzyme and the individual subunits in vitro and were associated with all the three subunits of CK2 in vivo.In conclusion, we have shown in the present study the results of the existence of phosphorylation and interaction between CK2 and Pdx-1.Further functional analysis relating to the phosphorylation and interaction also provided a mechanism for temporal and spatial regulation of Pdx-1.更多还原