臺灣博碩士論文加值系統

P-STM抗體是我們實驗室針對PAK2的自我磷酸化作用點，利用此抗原序列SKRSpTMVGTPY，製造出一個可辨認專一性磷酸化抗體 (Yu et al., Biochem. J. 334, 121-131, 1998; Tsai et al., J. Cell. Biochem. 94, 967–981, 2005)。它不僅可以辨認活化態/自我磷酸化的的PAK2，還可以對於處於分裂週期之A431與HeLa細胞蛋白萃取液中許多磷酸化蛋白都有辨識能力，而其中兩個磷酸化蛋白分別被鑑定為lamin A及lamin C。Lamins A/C已知在細胞分裂時期會被p34cdc2磷酸化。我們發現在試管中p34cdc2可對經由免疫沈澱分離的lamins A/C進行磷酸化作用，而且此一磷酸化作用可以產生P-STM抗體可辨認的磷酸化表位（epitope），所以我們推測P-STM抗體辨認Nocodazole處理過後(處於分裂週期)的lamins A/C磷酸化的表位可能是被p34cdc2作用的。我們實驗的目標是希望可以找出lamins A/C在細胞分裂時期被P-STM辨認的磷酸化表位及定量在不同時期lamins A/C磷酸化位點之磷酸化強度。利用質譜學及分子生物學的方法，我們成功的找到及定量lamins A/C在細胞分裂時期被P-STM辨認的磷酸化位置。實驗結果發現，在試管中p34cdc2是透過對lamin C之Thr-19磷酸化作用，而產生P-STM抗體的磷酸化表位。而進一步利用「穩定同位素標定胺基酸細胞培養」(Stable Isotope Labeling with Amino acids in Cell culture, SILAC)的方法也可以看出lamin C之12SGAQASS19TPLSPT25R片段訊號在有無處理nocodazole的細胞中有明顯的差異。藉由瞭解lamins A/C被P-STM辨認的磷酸化位置，我們希望可以利用此一線索找出更多有關細胞週期調節的磷酸化蛋白分子及其磷酸化位置。

P-STM, a phospho-epitope-specific antibody generated against the autophosphorylation/regulatory site of p21-activated kinase (PAK2) (antigen sequence: SKRSpTMVGTPY), not only recognizes the autophosphorylated/ activated PAK2 but also many phosphoproteins including lamins A/C in mitotic HeLa and A431 cells (Yu et al., 1998; Tsai et al., 2005). Lamins A/C are known to be phosphorylated by p34cdc2 kinase in mitotic cells. The P-STM recognizable epitopes on lamins A/C may serve as good targets to monitor lamins A/C phosphorylation levels during cell cycle. Lamins A/C immunoprecipitated from unsynchronized HeLa cells could be phosphorylated in vitro by p34cdc2 and this phosphorylation created epitope(s) on lamins A/C to P-STM, suggesting that the phosphorylation site(s) mediated by p34cdc2-catalyzed reaction in vitro may represent the one(s) recognized by P-STM in mitotic cells. The aims of this study are to identify the P-STM recognizable epitopes on lamins A/C in mitotic phase and to quantify the sites of lamins A/C differentially phosphorylated at interphase and mitotic phase. We applied mass spectrometry and molecular biology technologies to identify and quantify lamins A/C phosphorylation site(s) recognized by P-STM. We showed that Thr-19 of lamins C after phosphorylation by p34cdc2 kinase represented the sole epitope to P-STM. The different levels of lamin A/C peptide 12SGAQASS19TPLSPT25R in control and nocodazole-treated cells revealed by SILAC (Stable Isotope Labeling with Amino acids in Cell culture) method suggest that Thr-19 of lamins A/C is differentially phosphorylated at interphase and mitotic phase.

指導教授推薦書 iii
口試委員會審定書 iv
碩士論文電子檔案上網授權書 iii
長庚大學碩士紙本論文著作授權書 iv
誌謝 v
中文摘要 vi
CHAPTER I Introduction 1
CHAPTER II Material and methods 7
CHAPTER III Results 16
CHAPTER IV Discussion 24
Reference lists 24
Figure legends 35
Appendix 56

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