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系統識別號 U0026-0812200911510240
論文名稱(中文) Mammalian Ste20 kinase 3 (MST3) 之功能為抑制細胞爬行且此功能受其自體磷酸化及離子使用特性所影響
論文名稱(英文) Inhibition of migration by mammalian Ste20 kinase 3 (MST3) involves its autophosphorylation and metal ion requirement
校院名稱 成功大學
系所名稱(中) 基礎醫學研究所
系所名稱(英) Institute of Basic Medical Sciences
學年度 94
學期 2
出版年 95
研究生(中文) 陸德容
研究生(英文) Te-jung Lu
電子信箱 lutejung@yahoo.com.tw
學號 s5886104
學位類別 博士
語文別 中文
論文頁數 87頁
口試委員 口試委員-余兆松
召集委員-湯銘哲
口試委員-黃奇英
口試委員-劉校生
指導教授-賴明德
口試委員-蘇益仁
中文關鍵字 自體磷酸化  細胞爬行  離子使用特性 
英文關鍵字 Mammalian Ste20 kinase 3 (MST3)  autophosphorylation  metal ion requirement  migration 
學科別分類
中文摘要 我們clone到Ste20 serine/threonine kinase家族中的一員,由結構看來,它與mammalian ste20-like kinase(mst1/2)相似,故被命名為mammalian STE20-like kinase 3 (MST3)。MST3是一種serine/threonine(S/T)kinase,卻不尋常的利用錳離子當輔因子,但MST3是否可利用其他離子則尚未被有系統的分析過。在我們測試的離子中生理濃度下的錳、鈷、鎂、鋅皆可活化內生性、外生性及baculovirus 所表現的MST3。利用phosphoamino acid的分析,我們發現當錳、鈷或鎂當離子時,最主要的磷酸化位置是threonine,而鋅離子當輔因子時,serine及threonine均被磷酸化,且其磷酸化程度一樣強。據我們了解,這是第一篇除了內生性、外生性表現的S/T kinase、baculovirus 所表現的S/T kinase能被鋅離子活化的報導。另外MST3的生物功能除了已知與細胞凋亡與氧壓力有關外,其他則尚不清楚。故我們以RNAi的方式,將一株爬行較慢的MCF7細胞的MST3抑制後,發現MST3的功能是可抑制細胞伸出突觸及爬行。並且,我們將野生型的MST3送入另一株爬行較快的MDCK細胞,發現它能有效的抑制MDCK的突觸伸出及爬行。另外,我們也利用序列比對,trptic digestion,mass spectrometry,及定點突變等方式,證實Thr178即為MST3自體磷酸化的位置,並將T178A MST3,一個無法自體磷酸化的MST3送入MDCK細胞中,發現其喪失了MST3抑制MDCK細胞爬行的功能。在MCF7及MDCK細胞中均發現,即使細胞完全攤開後,有野生型HA-MST3表現時,pY31及pY118明顯較無MST3時高,故MST3能藉由維持paxillin之磷酸化而壓抑的細胞爬行。另外,我們將Q47突變成E後,發現Mn2+喪失活化MST3的能力,但Mg2+活化MST3的能力則無改變。送入Q47E-MST3的MDCK能抑制MST3的作用,使collective migration增快,但在單一MDCK的爬行速度上,只能部份抑制MST3的作用,pY31及pY118則介於parental MDCK與WT-MST3 MDCK間。本篇研究使我們了解MST3如何藉由自體磷酸化及利用2價陽離子Mn當輔因子來調控paxillin之磷酸化而控制細胞爬行的內部分子機制,並且發現MST3活化時,鋅離子可能扮演與其他離子不同之角色。
英文摘要 We cloned a novel human member of the Ste20 serine/threonine protein kinase using degenerate polymerase chain reaction against conserved catalytic subdomains of tyrosine kinases. Based on its domain structure, MST3 belongs to serine/threonine kinase, and is structurally close related to the mammalian STE20-like kinase (mst1/2), so is named as mammalian STE20-like kinase 3 (MST3). MST3 is a serine/threonine kinase with a unique preference for manganese ion as a cofactor in vitro. However, the metal ion cofactor preference for MST3 has not been systemically examined. Four metal ions (Mg+2, Mn+2, Zn+2, and Co+2) activate endogenous, exogenous, and baculovirus expressed recombinant MST3 within the physiological concentration range. Mn+2, Co+2, and Mg+2-dependent autophosphorylation of MST3 is mainly on threonine residue while Zn+2-stimulated MST3 autophosphorylation is on both serine and threonine residues. To our knowledge, this is the first report showing Zn+2 as the metal ion cofactor of a recombinant serine/threonine kinase. In addition, the biological function of MST3 is largely unknown. Suppression of endogenous MST3 by small interference RNA enhanced cellular migration in MCF7 cells with reduced expression of E-cadherin at the edge of migrating cells. The expression of surface integrin and Golgi apparatus were not altered, but phosphorylation on Tyrosine-118 and 31 of paxillin was decreased by MST3 RNAi expression. Threonine-178 was determined to be one of the two main autophosphorylation sites of MST3 in vitro by us. Mutant T178A-MST3 lost auto- phosphorylation and kinase activities. Overexpression of wild type MST3, but not the T178A mutant MST3, inhibited migration and spreading in MDCK cells. Q47E MST3 had reduced kinase activity using manganese ion as cofactor, but retained the same kinase activity in the presence of magnesium ion. The Q47E mutation abolished the effect of MST3 on collective migration, but only partially affected single-cell migration. The extent of pY118 of paxillin is correlated with migration behavior of MDCK cells expressing various forms of MST3. We conclude that MST3 regulates cell migration in a manner involving autophosphorylation, kinase activity, and metal ion cofactor and the distinct autophosphorylation pattern on MST3 suggests that MST3 may exert various types of kinase reactions depending on the type of metal ion cofactor used.
論文目次 中文摘要 I
英文摘要 III
誌謝 IV
目錄 V
圖目錄 VIII
表目錄 IX
第一章 背景 1
ㄧ Ste20 (Sterile 20) 族群蛋白激酶 1
二 MST 家族蛋白的功能 2
三 細胞爬行 4
四 金屬離子在激酶所扮演的角色 6
五 MST3在發育上之重要性 9
第二章 研究目標 10
第三章 結果 11
ㄧ 將MST3 cDNA從膀胱癌細胞中clone出來 11
二 MST3 mRNA廣泛分佈於各組織中 11
三 測試α-MST3-C’抗體的特異性 11
四 核醣核酸干擾作用降低細胞內MST3 的表現 12
五 細胞內MST3 降低表現並不影響細胞增生的速度 13
六 MST3的表現可抑制MCF7細胞的collective migration 13
七 MST3抑制的細胞爬行經由維持paxillin之磷酸化所造成,但不經由控
制integrin的表現或聚集而形成 14
八 確認in vitro baculovirus所表現的MST3可磷酸化peptide 178裡的
胺基酸的位置 15
九 Threonine 178是MST3自體磷酸化所必須的 16
十 將Threonine 178突變,可降低 MDCK細胞中MST3的自體磷酸化的
程度,酵素活性,及MST3抑制其爬行之功能 17
十一 Q47E-MST3可影響MST3對離子的喜好程度並可改變MST3對
MDCK collective migration之影響 18
十二 T178A,Q47E-MST3可影響MST3對MST3對MDCK細胞展開,單一細胞爬
行之速率及paxillin磷酸化之程度 18
十三 除了錳離子,其他離子也可當作輔因子來活化細胞內內生性的MST3 19
十四. 鋅或鈷可當作外生性HA-MST3的輔因子 19
十五 鋅或鈷離子可當作純化的重組蛋白MST3的輔因子 20
十六 各種不同的2價離子對MST3酵素反應參數之影響 20
十七 鋅離子可使MST3自體磷酸化 20
十八 以CaEDTA,一種針對鋅離子的螯合劑,可使MST3之活性消失 21
十九 鋅離子造成MST3的自體磷酸化的位置是在serine及threonine胺基酸上 21
二十 MST3在發育過程中所扮演的角色 22
第四章 材料與方法 23
DNA constructs 23
Cell culture and transfection 23
Western blot analysis 23
Construction and purification of baculovirusexpressed MST3 24
Immunoprecipitation and in vitro kinase assay 24
Evaluation of MST3 kinetic parameters 25
In vitro labeling and phosphoamino acid analysis 25
Cell culture and antibodies 26
RNA Interference (RNAi) 27
MST3 mutant plasmids 27
Wound-healing assays 27
Migration Assays 27
Immunoprecipitation and in vitro kinase assay 28
MST3 substrate assay and peptides 29
Phosphopeptide analysis 29
Flow Cytometry analysis 29
Immunofluorescence Microscopy 30
第五章 結論 31
第六章 討論 32
ㄧ MST3在細胞內之功能 32
二 MST3抑制細胞爬行之機制 34
三 MST3自體磷酸化對其功能之影響 37
四 離子之喜好對MST3功能之影響 38
a 錳離子之影響 38
b 鋅離子之影響 40
五 MST3在發育過程中所扮演的角色 42
六 MST3的研究的重要性、應用性及未來之研究方向 43
七 Diagram of inhibition of migration by MST3 involves it autophosphorylation and metal ion requirement. 45
參考文獻 46
圖 56
表 85
自述 86
發表的著作 87
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