指導教授：蔡明道臺灣大學：生化科學研究所陳聖文Chen, Eric Sheng-WenEric Sheng-WenChen2014-11-262018-07-062014-11-262018-07-062014http://ntur.lib.ntu.edu.tw//handle/246246/261642細胞週期檢查點激酶扮演穩定真核生物基因組的重要關鍵。因應DNA受損，酵母菌檢查點激酶Rad53的激活分為兩步。首先是由Rad9或Mrc1檢查點蛋白媒介上游Mec1檢查點激酶對於Rad53的起動磷酸化；而該起動磷酸化則會進一步導致Rad53激酶結構域活化環的自動激活磷酸化。然而，這些媒介起動磷酸化的檢查點蛋白是如何調節Mec1在Rad53那些特定胺基酸位點上產生磷酸化，而此又如何導致Rad53激活機制仍然知之甚少。本篇論文，我們使用定量質譜分析法研究Rad53因應細胞週期S期的烷基化DNA損傷所發生的逐步活化磷酸化；並發現，Rad9和Mrc1兩個媒介蛋白與Rad53氮端Mec1可磷酸化作用的四個蘇氨酸位點（亦為Rad53-SCD1絲氨酸穀氨醯胺/蘇氨酸穀氨醯胺群集結構域1）以及Rad53-FHA2結構域可以密切整合以產生最佳的Rad53起動磷酸化，以導引顯著的自動激活。在Rad9或Mrc1單獨作用的狀況下，所媒介Mec1對於Rad53整體磷酸化的位點與模式非常的相似，甚至包括以前未發現的Rad53-SCD1內的三重與四重蘇氨酸磷酸化。Rad53-SCD1結構域內的起動磷酸化效率與其存在的蘇氨酸位點數量息息相關。當可作用的蘇氨酸位點越少，該結構域則會變成一個較差的Mec1磷酸化標的，而此時便需要兩個媒介蛋白MRC1和RAD9同時作用以達成的足夠的起動磷酸化來促進顯著的自動激活。對磷酸化蘇氨酸有專一辨識作用的Rad53-FHA結構域，特別是FHA2，主要是透過與檢查點媒介蛋白結合來調節起動磷酸化，但似乎並沒有參與SCD1內的起動磷酸化所導致的自動激活。最後，我們的結果還發現，當SCD1內四個蘇氨酸位點全都突變為無法被磷酸化的丙氨酸時，Rad53活化大大降低，但並未完全消失。而該突變體內的殘餘Rad53活性則是取決於Rad9而不是Mrc1的作用。總括所有的發現，我們的研究結果提供了磷酸化位點集群和檢查點媒介蛋白如何可以參與在體內蛋白激酶級聯信號傳導調節的一個範例，同時也說明一個透過RAD9途徑而卻不需要倚賴SCD1起動磷酸化的Rad53自動激活機制。該研究也展示出以質譜進行深入分析細胞體內信息傳導分子機制的功效。The cell cycle checkpoint kinases play central roles in genome maintenance of eukaryotes. Activation of the yeast checkpoint kinase Rad53 involves Rad9 or Mrc1 adaptor-mediated phospho-priming by Mec1 kinase, followed by auto-activating phosphorylation within its activation loop. However, mechanisms of how these adaptors regulate priming phosphorylation of specific sites and how this then leads to Rad53 activation remain poorly understood. Here we use quantitative mass spectrometry to delineate the stepwise phosphorylation events in the activation of endogenous Rad53 in response to S phase alkylation DNA damage, and show that the two Rad9 and Mrc1 adaptors, the four N-terminal Mec1-target TQ sites of Rad53 (Rad53-SCD1), and the Rad53-FHA2 coordinate intimately for optimal priming phosphorylation to support substantial Rad53 auto-activation. Rad9 or Mrc1 alone can mediate surprisingly similar Mec1-target site phosphorylation patterns of Rad53, including previously undetected tri- and tetra-phosphorylation of Rad53-SCD1. Reducing the number of TQ motifs turns the SCD1 into a proportionally poorer Mec1 target, which then requires the presence of both Mrc1 and Rad9 for sufficient priming and auto-activation. The phosphothreonine-interacting Rad53-FHA domains, particularly FHA2, regulate phospho-priming by interacting with the checkpoint mediators, but do not seem to play a major role in the phospho-SCD1-dependent auto-activation step. Finally, mutation of all four SCD1 TQ motifs greatly reduces Rad53 activation, but does not eliminate it, and residual Rad53 activity in this mutant is dependent on Rad9 but not Mrc1. Altogether, our results provide a paradigm for how phosphorylation site clusters and checkpoint mediators can be involved in the regulation of signaling relay in protein kinase cascades in vivo, and elucidate an SCD1-independent Rad53 auto-activation mechanism through the Rad9 pathway. The work also demonstrates the power of mass spectrometry for in-depth analyses of molecular mechanisms in cellular signaling in vivo.Table of Contents 論文口試委員審定書 i Acknowledgement ii 中文摘要 iv English Abstract vi Table of Contents viii List of Figures xii List of Tables xvi Chapter 1. General Introduction 1 1-1. Cell Cycle Checkpoint 1 1-1.1. The regulation of cell cycle progression 1 1-1.2. The control mechanisms that ensure the DNA fidelity 2 1-1.3. Assembly of sensors, transducers, and effectors for checkpoint surveillance and signaling 3 1-1.4. The molecular checkpoint pathways associated with DNA replication stress and DNA damages 6 1-2. Roles of the SCD and FHA Domains in the Regulation of Checkpoint Signaling 10 1-2-1. The general introduction of SCD domain 10 1-2-2. The FHA domain: structure and ligand specificity 11 1-2-3. Roles of SCDs and FHAs in the regulation of Chk-2 like checkpoint kinases 14 Chapter 2. Quantitative Mass Spectrometric Analyses for Rad53 Hyperphosphorylation in Response to DNA Alkylation Damage and Replication Stress 18 2-1. Introduction 18 2-2. Materials and Methods 23 2-2-1. Yeast strains 23 2-2-2. Cell synchronization, MMS treatment, and preparation of spiked-in SILAC standard 24 2-2-3. Western blotting and immunoprecipitation 24 2-2-4. LC-MS/MS and data Analysis 25 2-3. Results 27 2-3-1. Optimization and evaluation of Rad53 immunoprecipitation by EL7E1 27 2-3-2. Quantitative MS analysis of endogenous Rad53 phosphorylation in response to S phase DNA damage 28 2-3-3. New phosphorylation sites, multi-sites, and estimated stoichiometry 37 2-4. Discussion 43 Chapter 3. Quantitative Mass Spectrometry for In-depth Analyses of Phospho-priming and Auto-activation Mechanisms of the Checkpoint Kinase Rad53 in Vivo 45 3-1. Introduction 45 3-2. Materials and Methods 48 3-2-1. Yeast strains 48 3-2-2. Western blotting and immunoprecipitation 48 3-2-3. Enrichment of phosphopeptides with TiO2 beads 48 3-2-4. LC-MS/MS and Data Analysis 49 3-2-5. Expression and purification of recombinant full-length Rad53 and Rad53-FHA1 domain 49 3-2-6. In vitro peptide pull-down assays 50 3-3. Results 51 3-3-1. Identification of kinase-dependent Rad53 phosphorylation events with the kinase-deficient rad53-K227A allele 51 3-3-2. Mrc1 and Rad9 function redundantly in Rad53 phosphorylation events 55 3-3-3. SCD1 phospho-priming is a key regulator but not sole determinant of Rad53 auto-activation 58 3-3-4. Rad9 but not Mrc1 also functions as a scaffold for SCD1-independent Rad53 auto-activation 61 3-3-5. Both Mrc1 and Rad9 adaptors are required for sufficient SCD1 priming phosphorylation when fewer SCD1-TQ motifs are preserved 62 3-3-6. Multiple SCD1 threonines are required for RAD53-dependent DNA damage survival in the absence of the Mrc1 or Rad9 adaptor 67 3-3-7. The FHA2 domain plays a major role in SCD1 priming phosphorylation 68 3-3-8. FHA1-pSCD1 binding is not the major regulator for Rad53 auto-activation 71 3-3-9. Novel roles of FHA1 and multi-phosphorylated SCD1 in Rad53-S424 phosphorylation and full-length Rad53 binding 72 3-4. Discussion 75 3-4-1. Possible molecular basis for the redundant functions of Mrc1 and Rad9 in the phospho-priming step 76 3-4-2. Coordination of Mec1 adaptors and Mec1-target sites for optimal priming of Rad53 by Mec1 77 3-4-3. Possible intermediate complexes of Rad53 auto-activation 78 3-4-4. Rad53-SCD1: a hotspot of Mec1 signaling control 79 3-4-5. Implication for the in vivo activation mechanisms of Rad53 and other Chk2-like kinases 80 References 82 Appendix 98 List of abbreviations 987605122 bytesapplication/pdf論文公開時間：2014/03/08論文使用權限：同意有償授權(權利金給回饋學校)檢查點激酶FHA結構域質譜Rad9/Mrc1檢查點媒介蛋白磷酸化Rad53激酶絲氨酸穀氨醯胺/蘇氨酸穀氨醯胺群集結構域(SCD)thesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/261642/1/ntu-103-D96B46001-1.pdf