前體信使核糖核酸（pre‐mRNA）的剪接是在RNA成熟與蛋白質多樣性發生中所必需的一類高度動態的過程。作為一類特定的非小核糖核蛋白剪接因子，絲氨酸精氨酸（SR）蛋白在mRNA的組成型剪接及選擇性剪接，mRNA的轉運與翻譯中均扮演關鍵角色。SR蛋白在其氮端含有1個或2個RNA識別基序（RRMs），其碳端的RS結構域含有連續排列且可被高度磷酸化的精氨酸絲氨酸（RS）二肽。SR蛋白的磷酸化水平可調節其亞細胞定位與生理功能，而屬於蛋白激酶超家族的SR蛋白激酶（SRPK）家族負責SR蛋白的磷酸化修飾。 / 在此項課題中，我們著重於SRPK2獨特的底物特異性及其磷酸化機制的研究。課題選用兩個代表不同類型的底物：人類絲氨酸精氨酸剪接因子1（SRSF1）和人類細胞凋亡染色質聚縮引導因子S（acinusS）。研究結果顯示，氮端非激酶區為SRPK2對SRSF1和acinusS的激酶活力所必需。另外，雖然兩種底物類型一級結構迥異，但一個位於SRPK2的大葉且保守的docking groove，負責對它們的識別與結合。 / SRPK1以processive機制催化SRSF1中8‐10個位點，而我們的實驗結果顯示SRPK2以processive機制磷酸化SRSF1的約5‐6個位點。我們證明，SRPK2的docking groove對processive機制的磷酸化有著重要作用，而且位於dockinggroove中的組氨酸601決定了SRPK2較低的processvity。有趣的是，SRPK2的docking groove也在acinusS絲氨酸422的位點特異性磷酸化中起關鍵作用。我們證明該位點特異的磷酸化機制主要是由SRPK2的docking groove與位於acinusS磷酸化位點氮端推定的docking motif之間的離子型相互作用，及其隨之與一個同樣位於acinusS的磷酸化位點N端負的電荷區域之間的離子型排斥作用所調節。 / 這些結果顯示，SRPK2的docking groove採取了兩種不同的磷酸化機制，因而其底物可以或者processive機制，或者高度位點特異的機制被磷酸化修飾。此外，為闡明此兩種迥異的磷酸化機制的分子基礎，蛋白質晶體學研究正在進行之中。 / Pre‐mRNA splicing is a highly dynamic process that plays an essential role in mRNA maturation and protein diversity generation. One particular family of non‐small nuclear ribonucleoproteins (snRNPs) splicing factors, the serinearginine (SR) proteins, play critical roles in both constitutive and alternative mRNA splicing, mRNA transport, and translation. N‐terminus of SR proteins consists one or two RNA recognition motifs (RRMs), and the C‐terminal RS domain contains continuous RS dipeptides that could be extensively phosphorylated. The phosphorylation states of SR proteins regulate their subcellular localization and physiological functions. SR protein kinase (SRPK) family is a member of the kinase superfamily that accounts for SR protein phosphorylation. / In this study, we focused on the distinct substrate specificity and phosphorylation mechanism of SRPK2. Two substrates representing different classes are selected: human serine/arginine splicing factor 1 (SRSF1) and human apoptotic chromatin condensation inducer in the nucleus S (acinusS). Our results showed that the N‐terminal non‐kinase region of SRPK2 is required for the full catalytic activity towards both SRSF1 and acinusS. Besides, a conserved docking groove in the large lobe of SRPK2 was shown responsible for the recognition and binding of both substrate classes despite the significant difference in their primary structures. / While SRPK1 modifies SRSF1 for 8‐10 sites in a processive manner, our results show that SRPK2 processively phosphorylates SRSF1 for approximately 5‐6 sites. We provided evidence that the docking groove of SRPK2 is important for the processive phosphorylation mechanism and His601 within the groove accounts for the lower processivity. Interestingly, the docking groove also plays a critical role in the site‐specific phosphorylation of acinusS at Ser422. We demonstrated that the single site phosphorylation mechanism of SRPK2 is mainly regulated by ionic interaction with a putative docking motif, and the following ionic repulsion between the docking groove and an electronegative region N‐terminal to the P‐site of acinusS. / These results suggest that the docking groove of SRPK2 adopts two distinct phosphorylation mechanisms so that different RS domains can be phosphorylated in either processive or highly site‐specific manner. Protein crystallography studies are undergoing to provide the molecular basis of the two distinct phosphorylation mechanisms. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Liang, Ning. / Thesis (Ph.D.) Chinese University of Hong Kong, 2014. / Includes bibliographical references (leaves 151-170). / Abstracts also in Chinese.
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_1077643 |
| Date | January 2014 |
| Contributors | Liang, Ning (author.), Ngo, Jacky Chi Ki (thesis advisor.), Chinese University of Hong Kong Graduate School. Division of Life Sciences, (degree granting institution.) |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, bibliography, text |
| Format | electronic resource, electronic resource, remote, 1 online resource (viii, 170 leaves) : illustrations (some color), computer, online resource |
| Rights | Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/) |
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