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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.

Thesis (Ph. D.)--University of Virginia, 2006. / Includes bibliographical references. Also available online through Digital Dissertations.

Thesis (Ph. D.)--Cornell University, January, 2009. / Vita. Includes bibliographical references (leaves 102-118).

Thesis (Ph. D.)--University of Washington, 1999. / Vita. Includes bibliographical references (leaves [157]-179).

SR蛋白在前體信使核糖核酸(pre-mRNA)的組成性剪接和選擇性剪接中扮演者重要的角色，在這個過程中它需要被SR蛋白激酶(SRPK) 燐酸化才能正常行使功能。經典的SR蛋白是由N端一到二個RNA識別基序(RRM) 以及C端一串精氨酸－絲氨酸(RS) 二肽所構成。SR蛋白的燐酸化調控它的亞細胞定位以及生理功能。此外，SR 蛋白激酶1(SRPK1) 和SR蛋白原型ASF/SF2的復合物結構顯示底物的結合需要第二個非標準的RRM結構域以及在N端可以被燐酸化的RS結構域，但是，第一個標準的RRM結構域對於SR 蛋白激酶1的結合卻是可以或缺的。 / 在這裡，我們展示了SR蛋白激酶2(SRPK2) 結合並且燐酸化SRp20的RS結構域，SRp20是另外一個只包含一個RNA識別基序(RRM) 的SR蛋白。與ASF/SF2相似的是，SRp20中的標準RNA識別基序對於SRPK2的結合並不是必要的。與此同時，我們發現錨定槽對於底物的識別作用在SRPK2中也是保守的，因為，錨定槽中四個關鍵氨基酸的突變會削弱它對SRp20的結合。 / 此外，現在認為SRPK2的功能已經不限於對前體信使核糖核酸(pre-mRNA) 的剪接調控。最近發現，SRPK2也可以燐酸化Tau蛋白並且介導阿爾茨海默疾病中的認知性缺陷。組成性的激活是SR蛋白激酶中的一個固有特性，然而人們對於它的調控機制還不是很清楚。因此, 為了更好的瞭解SRPK2，我們采用酵母雙雜交的方法並且最終發現一個新的SRPK2相互作用蛋白: ZNF187。 / ZNF187是一個可以結合血清反應元件(SRE) 的轉綠因子。我們的研究發現，它可以正向調控SRE的轉錄激活。然而，SRPK2在EGF的刺激下卻起着抑制的效果，其中EGF的刺激會促使SRPK2進入細胞核。進一步證實，通過RNAi干擾的方法敲掉SRPK2可以增加ZNF187誘導的SRE活性。在共轉染實驗中，SRPK2可以把ZNF187誘導的SRE活性逆轉到本底水平。對於可以和EGF刺激的SRPK2有着相似細胞定位的缺失或者突變研究發現，它們都可以產生相一致的抑制現象。於此相反，對於和SRPK2有着不同細胞定位的突變，它卻不能產生抑制效果。因此，我們認為在EGF的刺激下，SRPK2進入細胞核並且負向的調控ZNF187激活的SRE。令人驚訝的是，如果細胞在FBS的刺激下，SRPK2卻上調SRE活性，並且它可以協同增加ZNF187對於SRE的誘導。這些結果表明SRPK2對於ZNF187誘導的SRE轉綠調控是刺激物依賴的。 / SR proteins are critical players in regulating both constitutive and alternative pre-mRNA splicing, during which the phosphorylation by SR Protein Kinases (SRPKs) is required. Classical SR proteins contain one or two RNA Recognition Motifs (RRM) in their N terminus and a stretch of Arginine-Serine (RS) dipeptides in C terminus. Phosphorylation status of SR proteins regulates their subcellular localization as well as physiological function. In addition, complex structure of SRPK1 with ASF/SF2, a prototype of SR protein, shows that substrate binding requires non-canonicalRRM2 domain and RS domain, which can be extensivelyphosphorylated. However, the canonical RRM1 domain is dispensable for such interaction. / Here we show that SRPK2 binds and phosphorylates SRp20, a classical single RRM domain-containing SR protein, at its RS domain. Similarly with ASF/SF2, the canonical RRM domain of SRp20 is dispensable for interacting with SRPK2. Meanwhile, we also find that a docking groove that iscritical for substrate binding in SRPK1 is also conserved in SRPK2, since mutations on four key residues in docking groove impair its binding affinity with SRp20. / In addition, SRPK2 is now known to function more then regulating mRNA splicing, such as cell proliferation and cell apoptosis. Recently, SRPK2 is also shown to be a kinase phosphorylating Tau and mediate the cognitive defects in Alzheimer’s disease (AD). Besides, an intrinsic character of SRPKs lies in that they are constitutively active, but the regulation mechanism is not well understood. Therefore, in order to obtain a better recognition about SRPK2, we applied yeast two-hybrid assay and eventually anew interaction partner called ZNF187 was identified. / ZNF187 is a transcriptional factor that binds with Serum Response Element (SRE). Our studies showed that it isa positive regulator of SRE activity. However, SRPK2 showed inhibiting effect on SRE activation with the treatment of EGF, which could induce its nucleus entry, when co-transfected, it reversed the stimulating effect on SRE by ZNF187 to basal level. Furthermore, knockdown of SRPK2 by RNAi would enhance ZNF187-stimuated SRE activation. Studies on truncation and mutations that have the similar effect with EGF-induced subcellular localization of SRPK2 also generated the same inhibiting phenomenon. In contrast, mutant that has distinct localization with SRPK2 wild type failed to exert suppression. Therefore, we conclude that with the treatment of EGF, SRPK2 moves into nucleus and negatively regulates ZNF187-stimulated transactivation of SRE. Surprisingly, when cells were treated with FBS, SRPK2 showed stimulation on SRE activity and it synergized ZNF187-stimulated effect on SRE, indicating that transcriptional regulation of SRPK2 on ZNF187-stimulated SRE activity is stimuli-dependent. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Shang, Yong. / Thesis (Ph.D.) Chinese University of Hong Kong, 2014. / Includes bibliographical references (leaves 113-137). / Abstracts also in Chinese.

Li, Man Wah. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 109-119). / Abstracts in English and Chinese. / Thesis Committee --- p.I / Statement --- p.II / Abstract --- p.III / 摘要 --- p.V / Acknowledgements --- p.VI / Abbreviations --- p.VIII / Abbreviations of Chemicals --- p.X / List of Tables --- p.XI / List of Figures --- p.XII / Table of Contents --- p.XIII / Chapter Chapter 1 --- Literature Review --- p.1 / Chapter 1.1 --- General amino acid control in yeast --- p.1 / Chapter 1.2 --- Mammalian eIF2α kinases --- p.7 / Chapter 1.2.1 --- Heme-regulated inhibitor kinase (EIF2AK1/HRI) --- p.7 / Chapter 1.2.2 --- Protein kinase dsRNA-dependent (EIF2AK2/PKR) --- p.8 / Chapter 1.2.3 --- PKR-like ER kinase (EIF2AK3/PERK) --- p.9 / Chapter 1.2.4 --- General control non-repressible 2 (EIF2AK4/GCN2) --- p.10 / Chapter 1.2.5 --- Activating transcription factor 4 (ATF4) --- p.11 / Chapter 1.3 --- Plant General Amino Acid Control --- p.12 / Chapter 1.3.1 --- Studies of the homolog of GCN2 in Arabidopsis thaliana --- p.12 / Chapter 1.3.2 --- Studies of the homolog of other eIF2a kinase in plant --- p.14 / Chapter 1.3.3 --- Studies of the homolog of other GAAC components --- p.14 / Chapter 1.4 --- Previous works in our lab --- p.15 / Chapter 1.5 --- Hypothesis and Objectives --- p.17 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- Materials --- p.18 / Chapter 2.1.1 --- "Bacterial cultures, plant materials and vectors" --- p.18 / Chapter 2.1.2 --- Primers --- p.21 / Chapter 2.1.3 --- Commercial kits --- p.25 / Chapter 2.1.4 --- "Buffer, solution, gel and medium" --- p.25 / Chapter 2.1.5 --- "Chemicals, reagents and consumables" --- p.25 / Chapter 2.1.6 --- Enzymes --- p.25 / Chapter 2.1.7 --- Antibodies --- p.25 / Chapter 2.1.8 --- Equipments and facilities --- p.25 / Chapter 2.2 --- Methods --- p.26 / Chapter 2.2.1 --- Growth conditions of Arabidopsis thaliana --- p.26 / Chapter 2.2.1.1 --- Surface sterilize of Arabidopsis thaliana seed --- p.26 / Chapter 2.2.1.2 --- Growing of Arabidopsis thaliana --- p.26 / Chapter 2.2.1.3 --- Treatment of Arabidopsis seedling --- p.26 / Chapter 2.2.2 --- Basic molecular techniques --- p.27 / Chapter 2.2.2.1 --- Liquid culture of Escherichia coli --- p.27 / Chapter 2.2.2.2 --- Preparation of plasmid DNA --- p.27 / Chapter 2.2.2.3 --- Restriction digestion --- p.27 / Chapter 2.2.2.4 --- DNA purification --- p.28 / Chapter 2.2.2.5 --- DNA gel electrophoresis --- p.28 / Chapter 2.2.2.6 --- DNA ligation --- p.29 / Chapter 2.2.2.7 --- CaCl2 mediated E. coli transformation --- p.29 / Chapter 2.2.2.8 --- Preparation of DNA fragment for cloning --- p.29 / Chapter 2.2.2.9 --- PCR reaction for screening positive E. coli transformants --- p.30 / Chapter 2.2.2.10 --- DNA sequencing --- p.30 / Chapter 2.2.2.11 --- RNA extraction from plant tissue with tRNA --- p.31 / Chapter 2.2.2.12 --- Extraction of RNA without tRNA --- p.31 / Chapter 2.2.2.13 --- cDNA synthesis --- p.32 / Chapter 2.2.2.14 --- SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) --- p.33 / Chapter 2.2.2.15 --- Western blotting --- p.33 / Chapter 2.2.3 --- Sub-cloning of AtGCN2 --- p.34 / Chapter 2.2.3.1 --- Sub-cloning full length AtGCN2 into pMAL-c2 --- p.36 / Chapter 2.2.3.2 --- Sub-cloning of the N-terminal sequence of AtGCN2 into pMAL-c2 --- p.38 / Chapter 2.2.3.3 --- Sub-cloning of the C-terminal sequence of AtGCN2 into pMAL-c2 --- p.38 / Chapter 2.2.4 --- Cloning of the eIF2α candidates for the in vitro assay --- p.41 / Chapter 2.2.4.1 --- Cloning of At2g40290 (putative eIF2α candidate) --- p.41 / Chapter 2.2.4.2 --- Cloning of At5g05470 (putative eIF2α candidate) into pBlueScript KS II + --- p.43 / Chapter 2.2.4.3 --- Sub-cloning of At5g05470 into pGEX-4T-1 --- p.43 / Chapter 2.2.4 --- Expression and purification of fusion proteins --- p.45 / Chapter 2.2.5 --- Expression of fusion proteins in E. coli --- p.45 / Chapter 2.2.5.2 --- Extraction of E. coli soluble proteins --- p.45 / Chapter 2.2.5.3 --- Purification of GST tagged fusion protein --- p.46 / Chapter 2.2.5.4 --- Purification of MBP tagged fusion protein --- p.46 / Chapter 2.2.5.5 --- Concentration of purified fusion proteins --- p.46 / Chapter 2.2.5.6 --- MS/MS verification of purified fusion proteins --- p.47 / Chapter 2.2.6 --- Gel mobility shift assay --- p.47 / Chapter 2.2.6.1 --- Synthesis of short biotinylated RNA --- p.47 / Chapter 2.2.6.2 --- Ligation of short biotinylated RNA with tRNA --- p.48 / Chapter 2.2.6.3 --- Gel mobility shift assay --- p.48 / Chapter 2.2.6.4 --- Blotting of the sample on to nitrocellulose membrane --- p.48 / Chapter 2.2.6.5 --- Detection of the tRNA on the membrane --- p.49 / Chapter 2.2.6.6 --- Detection of the MBP fusion proteins on the membrane --- p.49 / Chapter 2.2.7 --- In vitro kinase assay of AtGCN2 --- p.49 / Chapter 2.2.8 --- In vitro translation inhibition assay --- p.50 / Chapter 2.2.8.1 --- In vitro transcription of HA mRNA --- p.50 / Chapter 2.2.8.2 --- In vitro translation --- p.51 / Chapter 2.2.8.3 --- Detection of the protein dot blot --- p.51 / Chapter 2.2.9 --- Gene expression analysis by real time PCR --- p.52 / Chapter 2.2.10 --- Total seed nitrogen analysis --- p.53 / Chapter Chapter 3 --- Results / Chapter 3.1 --- Blast search results suggested that AtGCN2 may be the sole eIF2α kinase in Arabidopsis thaliana --- p.54 / Chapter 3.2 --- Existence of two eIF2α candidates in Arabidopsis thaliana genome --- p.59 / Chapter 3.3 --- Fusion proteins were successfully expressed and purified --- p.63 / Chapter 3.4 --- C-terminal of AtGCN2 has a higher affinity toward tRNA than rRNA --- p.67 / Chapter 3.5 --- Both eIF2α candidates can be phosphorylated by full length AtGCN2 in vitro --- p.70 / Chapter 3.6 --- AtGCN2 can inhibit translation in vitro --- p.72 / Chapter 3.7 --- Overexpression of AtGCN2 did not affect expression of selected genes --- p.74 / Chapter 3.8 --- Overexpression of AtGCN2 did not affect seed nitrogen content and C:N ratio under normal growth conditions --- p.83 / Chapter Chapter 4 --- Discussion --- p.85 / Chapter 4.1 --- Existing evidence supported that AtGCN2 is the sole eIF2α kinase in Arabidopsis thaliana --- p.85 / Chapter 4.2 --- Kinase activities of AtGCN2 and its two substrates in Arabidopsis --- p.86 / Chapter 4.3 --- C-terminal binds tRNA in the gel mobility shift assay --- p.88 / Chapter 4.4 --- Overexpression of AtGCN2 did not affect gene expression of the transgenic lines under nitrogen starvation and azerserine treatment --- p.90 / Chapter 4.5 --- Overexpression of AtGCN2 did not alter the seed nitrogen content --- p.91 / Chapter 4.6 --- Existence of GCN4 and ATF4 in plant --- p.92 / Chapter 4.7 --- Alternative model without GCN4 and ATF4 homolog --- p.93 / Chapter 4.8 --- Possible application of the in vitro kinase assay --- p.94 / Chapter 4.9 --- Possible application of the in vitro translation inhibition analysis platform in future study --- p.95 / Chapter Chapter 5 --- Conclusion and Future Prospective --- p.97 / Appendices / Appendix I Commercial kits used in this project --- p.98 / "Appendix II Buffer, solution, gel and medium" --- p.99 / "Appendix III Chemicals, reagents and consumables" --- p.102 / Appendix IV Enzymes --- p.103 / Appendix V Antibodies --- p.104 / Appendix VI Equipments and facilities --- p.105 / Appendix VII Supplementary Data --- p.106 / Appendix VIII Amplification efficiency of real time primers --- p.108 / References --- p.109

PknB is a putative transmembrane eukaryotic-type serine/threonine protein kinase (STPK) in the cariogenic bacterium Streptococcus mutans that affects biofilm formation, genetic competence and acid tolerance. PknB contains extracellular penicillin-binding and serine/threonine kinase associated (PASTA) domains predicted to bind the D-alanyl-D-alanine (D-ala-D-ala) dipeptide of unlinked peptidoglycan. D-ala-D-ala elicits responses dependent and independent of the presence of pknB. Biofilm-derived cells of a pknB-deficient mutant (PKNB) exhibited concentration-dependent growth enhancement with D-ala-D-ala, which was not a nutrient response as addition of L-alanine or D-alanine did not give the same results. A total of 77 genes were differentially expressed in PKNB, including 7 with putative functions in fatty acid biosynthesis. PKNB was more sensitive to cell wall- and membrane-targeting antibiotics compared to wild-type. Based on these results, PknB in S. mutans appears to play an important role in cell wall biosynthesis, response to membrane stress and/or regulation of cell membrane composition.

PknB is a putative transmembrane eukaryotic-type serine/threonine protein kinase (STPK) in the cariogenic bacterium Streptococcus mutans that affects biofilm formation, genetic competence and acid tolerance. PknB contains extracellular penicillin-binding and serine/threonine kinase associated (PASTA) domains predicted to bind the D-alanyl-D-alanine (D-ala-D-ala) dipeptide of unlinked peptidoglycan. D-ala-D-ala elicits responses dependent and independent of the presence of pknB. Biofilm-derived cells of a pknB-deficient mutant (PKNB) exhibited concentration-dependent growth enhancement with D-ala-D-ala, which was not a nutrient response as addition of L-alanine or D-alanine did not give the same results. A total of 77 genes were differentially expressed in PKNB, including 7 with putative functions in fatty acid biosynthesis. PKNB was more sensitive to cell wall- and membrane-targeting antibiotics compared to wild-type. Based on these results, PknB in S. mutans appears to play an important role in cell wall biosynthesis, response to membrane stress and/or regulation of cell membrane composition.