本文選題：磷酸化　切入點：蛋白質(zhì)相互作用　出處：《華中科技大學(xué)》2010年博士論文

【摘要】： 蛋白質(zhì)的磷酸化修飾是生物體內(nèi)最重要翻譯后修飾(post-translational modification)方式之一,在哺乳動物細(xì)胞中,至少有1／3的蛋白質(zhì)發(fā)生過磷酸化,因而其作用幾乎涵蓋了所有的生理及病理過程,尤其在細(xì)胞的信號傳遞過程中占有及其重要的地位。蛋白質(zhì)的磷酸化過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。人類基因組約2%的基因編碼了518種蛋白激酶5,作為最早被鑒定出的且最為重要的蛋白激酶之一,酪蛋白激酶2(casein kinase 2, CK2)是一種高度保守的、在各種真核生物中都廣泛存在,并且具有多種生理功能的絲氨酸／蘇氨酸激酶。CK2是由兩個催化亞基(α和α')和兩個調(diào)節(jié)亞基(β)組成的異四聚體(α2β2,α'2β2或αα'β2)；其廣泛的參與了細(xì)胞正常功能的調(diào)控,如基因表達(dá),蛋白合成,細(xì)胞周期調(diào)控等,同時還參與如癌癥發(fā)生,抗凋亡等病理過程。2003年已報道CK2有多達(dá)307種作用底物,其中約五分之一的底物為調(diào)節(jié)基因表達(dá)的轉(zhuǎn)錄因子。 胰十二指腸同源盒-1(Pancreatic duodenal homeobox-1, Pdx-1)是由Hox樣同源盒基因編碼的轉(zhuǎn)錄因子。Pdx-1是參與包括胰島素基因在內(nèi)的多個β細(xì)胞特異性基因轉(zhuǎn)錄的主控轉(zhuǎn)錄因子。生理方面,Pdx-1參與了胰腺的發(fā)育和分化,促進(jìn)胰島β細(xì)胞增殖、成熟和功能的維持；病理方面,除了Pdx-1的基因變異與糖尿病的發(fā)生、發(fā)展密切相關(guān)外,還發(fā)現(xiàn)Pdx-1在包括胰腺癌、結(jié)腸癌、肺癌和乳腺癌在內(nèi)的多種腫瘤細(xì)胞中呈現(xiàn)過表達(dá),且認(rèn)為Pdx-1在腫瘤細(xì)胞中有促增殖效應(yīng),可能與腫瘤的發(fā)生、發(fā)展、轉(zhuǎn)移及對化療的耐藥有關(guān)。鑒于Pdx-1的功能如此關(guān)鍵,闡明其在細(xì)胞內(nèi)表達(dá)和激活的分子調(diào)控機制已成為多個研究領(lǐng)域內(nèi)的一個熱點問題。 近幾年越來越多的研究表明,Pdx-1為細(xì)胞內(nèi)可磷酸化蛋白,且翻譯后磷酸化修飾是調(diào)節(jié)Pdx-1活性的一個重要方式。本研究通過體內(nèi)、外磷酸化實驗證明,CK2激酶可磷酸化Pdx-1蛋白以及通過體外定點突變技術(shù)尋找到了Pdx-1蛋白的磷酸化位點即位于其羧基末端的thr231和ser232。并進(jìn)一步通過熒光素酶活性分析實驗及蛋白合成抑制劑放線菌酮(CHX)和蛋白酶體抑制劑MG132實驗發(fā)現(xiàn),CK2激酶磷酸化Pdx-1后不僅可抑制其對胰島素基因的轉(zhuǎn)錄活性；而且可影響其自身蛋白的穩(wěn)定性,使其更易于受蛋白酶體依賴性(proteasome-dependent)蛋白降解途徑的降解；且該作用可能是導(dǎo)致抑制其轉(zhuǎn)錄活性的原因之一。另一方面,采用免疫熒光實驗,通過激光共聚焦顯微鏡我們發(fā)現(xiàn),雖然CK2激酶的磷酸化作用并未影響Pdx-1蛋白的亞細(xì)胞定位；但是Pdx-1與CK2激酶各亞基(α,α',β)熒光圖片在細(xì)胞核內(nèi)的重疊表明他們在核內(nèi)共定位,因而提示Pdx-1與CK2激酶可能存在分子間相互作用。體外利用GST Pull-down、Far-western實驗和在體內(nèi)用免疫共沉淀實驗分別進(jìn)一步證明了Pdx-1蛋白可與CK2激酶各亞基和全酶發(fā)生特異性結(jié)合。綜上所述,本研究從分子、細(xì)胞水平上提供了CK2激酶和Pdx-1蛋白磷酸化和相互作用的實驗依據(jù),并進(jìn)一步探討了該作用的重要生物學(xué)意義,有助于我們理解Pdx-1在參與細(xì)胞事件中的空間和時間調(diào)控機制。
[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.

【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2010
【分類號】：R363

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相關(guān)期刊論文 前2條

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2 Nikiforos Ballian;Francis Charles Brunicardi;;Transcription factor PDX-1 in human colorectal adenocarcinoma:A potential tumor marker?[J];World Journal of Gastroenterology;2008年38期

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本文編號：1692829