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研究生:趙東仁
研究生(外文):Tong-Zang Chao
論文名稱:磷酸化對人類securin功能的影響
論文名稱(外文):Effects of phosphorylation on the functions of hSecurin
指導教授:陳紀雄
指導教授(外文):Ji-hshiung Chen
學位類別:碩士
校院名稱:慈濟大學
系所名稱:分子生物及細胞生物研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
畢業學年度:96
語文別:英文
論文頁數:49
中文關鍵詞:磷酸化細胞週期癌症轉移
外文關鍵詞:hSecurinphosphorylationcell cycleMAPKmetastasis
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人類的securin是腦垂腺腫瘤變形基因的同一產物。正常情況下,
hSecurin 在細胞週期的姐妹染色體分裂其間扮演了一個重要的角
色。在intrphase,hSecurin 是seperase 的抑制者,當細胞進入
mitosis/anaphase,hSecurin 會被anaphase-promoting complex
(APC) 分解。而之前的microarray研究顯示出hSecurin在會轉移的
細胞中有過度表現的情形發生,因此,我們檢驗乳癌細胞株其中會轉
移的MDA-MB-231和不會轉移的MCF-7裡hSecurin的表現,結果發現
在MDA-MB-231 中hSecurin 有過度表現。於是我們推測hSecurin 的
過度表現是由於不正常的分解所造成,因此我藉由在此兩種細胞內加
入cycloheximide 的實驗中來探討hSecurin 的分解,我們的實驗結
果顯示出MCF7中hSecurin的分解要比在MDA-MB-231中更快。
在分解的過程中hSecurin會先被磷酸化後才會被ubquitinated,
最終導致分解。早期的研究證明MAP kinase會磷酸化大鼠的securin
(Ser162),而和MAP kinase 相同的recognition motif(PPS165P)也在
人類的securin中被發現。因此,我們試著利用in vitro kinase assay
去證明MAP kinase可以磷酸化hSecurin。接著我們突變掉hSecurin
上MAP kinase 可能會磷酸化的這個位置(Ser165)去證明突變後的
hSecurin 是不會被MAP kinase 所磷酸化,爾後我們會探討在
interphase此磷酸化位置和hSecurin的分解之間有何關聯性。
此外,hSecurin被APC分解是由於KEN box和destruction box (D
box)這兩段consensus sequences所造成,APC透過KEN box辨識出
hSecurin然後將ubiquitin接合在hSecurin的D box上,最後造成
hSecurin被26S proteasome分解。因此,我們試著突變掉單一或兩
者藉此探討這兩段consensus sequences 和hSecurin 的分解之間的
關係。除了上述之外,我們還要藉由wound healing 和transwell
assay 證明hSecurin 的磷酸化和分解是否會影響它在細胞移動和侵
入這兩方面所扮演的角色。
我們的實驗結果顯示出hSecurin 能被MAP kinase 磷酸化。將
hSecurin 上能被MAP kinase 磷酸化的位置突變掉(S165A),則
hSecurin能更穩定的存在於細胞中。此突變的hSecurin分解的情形
和在MDA-MB-231中hSecurin分解的情形恰好相似,而 hSecurin的
穩定度增高會造成轉移的細胞中hSecurin有過度表現的現象。
Human securin is identical to the product of the gene called human
pituitary tumor-transforming gene 1(hPTTG1). Normally, hSecurin plays
an important role in cell cycle during sister chromatids separation. At
interphase, hSecurin is an inhibitor of separase. When cells enter
mitosis/anaphase, hSecurin is degraded by anaphase-promoting complex
(APC). A previous microarray study showed that hSecurin was
over-expressed in most metastatic cells. We therefore examined the
expression of hSecurin in metastatic and non-metastatic breast cancer cell
lines, MDA-MB-231 and MCF-7 and found that hSecurin was
overexpressed in MDA-MB-231. Overexpression of hSecurin may be due
to abnormal degradation of hSecurin. Therefore we explored degradation
of hSecurin in MDA-MB-231 and MCF-7 by treating cells with
cycloheximide for various durations. Our results showed that hSecurin in
MCF7 cells were degraded more rapidly than that of MDA-MB-231 cells.
During the degradation process, hSecurin has to be phosphorylated then
ubquitinated and subsequently degraded. Early study showed that MAP
kinase can phosphorylate rat securin (Ser162) at interphase in murine cells.
A recognition motif (PPS165P) of MAP kinase is also found in human
securin. Therefore, we examined whether hSecurin could be
phosphorylated by MAP kinase by using in vitro kinase assay. Next, we
mutated MAP kinase phosphorylation site (Ser165) of hSecurin and
examined the relationship between this phosphorylation site and
degradation of hSecurin at interphase. In addition, the degradation of
hSecurin by APC is due to two consensus sequences, KEN box and
destruction box (D box). Through recognition KEN box on hSecurin,
APC can ligate ubiquitin on D box of hSecurin and subsequently the
hSecurin is degradation by 26S proteasome. Therefore, we also mutated
KEN box、D box or both and explored the relationship between two
consensus sequence and degradation of hSecurin. Futhermore, we
examined whether the phosphorylation and degradation can affect
hSecurin’s role on cell migration and invasion by wound healing and
transwell assay. Our results showed that hSecurin can be phosphorylated
by MAP kinase in vitro and in vivo, and the mutated MAP kinase
phosphorylation site of hSecurin (S165A) was more stable than wild type
hSecurin in cells. The degradation of this mutant hSecurin was similar to
its degradation in MDA-MB-231. The stable hSecurin caused
overexpression of hSecurin in metastatic cells.
Abstract ………………………………………………I
中文摘要 ………………………………………………III
Content ………………………………………………IV
Introduction……………………………………………1
Materials ………………………………………………8
Methods ………………………………………………12
Results ………………………………………………17
Discussion …………………………………………23
References ……………………………………………27
Appendix ……………………………………………31
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