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研究生(外文):Cuei-Wen Chen
論文名稱(外文):The Structure basis of FAK-derived phosphotyrosine-peptide bound to Grb2-SH2 domain
指導教授(外文):Ming-Tsan SuChun-Hua Hsu
中文關鍵詞:FAK磷酸化胜肽Grb2-SH2核磁共振圓二色光譜X-ray 結晶繞射法
外文關鍵詞:FAKphosphopeptideGrb2-SH2NMRCDX-ray crystallography
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Focal adhesion kinase (FAK)是一個細胞內的酪胺酸磷酸酶,它活化後會產生一連串細胞的生理反應,包括細胞的吸附、遷移、入侵、增生和存活。在數種癌症中,常會見到FAK 的過量表現,因此FAK 有潛力作為一個抗癌的藥物標的。
FAK 上酪胺酸的磷酸化對訊息傳遞的調控扮演重要的角色,其中一個在FAT domain 上第925 個胺基酸酪胺酸(Tyrosine),位於螺旋1 上,存在有Grb2-SH2 domain 的結合序列pYXNX,當Y925 磷酸化後,會與Grb2-SH2 結合,接著活化Ras-MAPK 的訊息傳遞路徑,且Y925 的磷酸化與促進腫瘤形成的血管增生有關。
多數的SH2 domain 會和延伸的胜肽結合,但Grb2-SH2 內Trp 的側鏈會防止它和延伸的胜肽卻和形成β-turn 構形的胜肽結合,然而FAT 上的Y925 卻位於α 螺旋。本研究想了解是否FAT 上的螺旋1 在與Grb2-SH2 結合後產生構形的變化為何?如此便能更清楚此兩個重要分子的結合模式及調控情形,未來或能設計抑制劑阻斷此一結合。
本篇利用圓二色光譜及核磁共振的方法來研究Grb2-SH2 及FAT衍生的胜肽pY925 其結合時構型的變化。圓二色光譜的結果顯示pY925 胜肽加入TFE 之後,會有螺旋的二級結構出現,而再加入Grb2-SH2 後,pY925 胜肽構型會有明顯的二級結構變化,且二維及三維核磁共振的結果顯示pY925 胜肽與Grb2-SH2 結合後化學位移劇烈變化的胺基酸與pY925/Grb2-SH2 的複合物晶體所解出的胜肽結合模式(β-turn)的變化一致,因此推論pY925 在與Grb2-SH2 結合時,FAT 會有構型的變化。

Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that plays a central role in signal transduction pathways that are initiated at sites of integrin-mediated cell adhesions and by growth factor receptors. FAK functions in the control of cell survival, proliferation, migration and invasion, and overexpression of FAK is found in many forms of cancer. Therefore FAK might be a potential target for anticancer drug development.
Tyrosine phosphorylation of FAK plays a significant role in regulating FAK-mediated signaling. One of the FAK phosphorylation sites is at tyrosine Y925 in human which located in the FAT domain.Y925 exists in a consensus Grb2-SH2 domain binding site pYXNX , and has been shown to bind to Grb2-SH2 domain when Y925 is phosphorylated. The FAT-Grb2-SH2 interaction activates the Ras/MAPK signaling pathway, and this linkage is essential in promoting angiogenesis.
Grb2 SH2 prefers a typical β-turn conformation for targeted motif binding because of steric hindrance caused by a bulky side chain of Trp 121, however, Y925 is located at helix1 of the four helix bundle in the FAT domain. The goal of this study is to determine the necessary of conformational change of FAK derived phosphopeptide bound to Grb2-SH2.
Thus, NMR (nuclear magnetic resonance) and CD (circular dichroism) spectroscopy and X-ray diffraction method were utilized to elucidate the peptide adaptation. The FAK phosphopeptide [NDKV(pY)ENVTG] in TFE solvent reveals the helical property as that presented in FAT domain. Interestingly, the CD spectra of FAK peptide bound to Grb2-SH2 in different molar ratio show beta-structure and helical conformation exchange. In addition, the structure of Grb2-SH2 complexed with FAK peptide was determined by heteronuclear multidimensional NMR and X-ray diffraction. Those data indicate the chemical shift-perturbed region on the domain is not linear but bent. Our NMR data, along with CD experiments suggest that phosphorylation of Y925 and subsequent Grb2 binding require FAT domain to undergo conformational change.

致謝 ii
摘要 iii
Abstract iv
目錄 v
圖目錄 vi
表目錄 vii
英文縮寫表 viii
壹、前言 1
貳、材料與方法 4
參、結果 17
一、蛋白表現載體之構築 17
二、蛋白表現及純化 17
三、 pY925 及與Grb2‐SH2 結合後的二級結構 18
四、Grb2‐SH2 和pY925 的結合能力 20
五、Grb2‐SH2 與pY925 的複合物結構分析 20
六、Grb2‐SH2 與pY925 複合物的結晶實驗 21
肆、討論 23
伍、圖表 25
陸、參考文獻 47
柒、附錄 51

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