ZAK 是含serine/threonine 的 Mixed-lineage kinase 一族，被歸類為MAP3Ks。ZAK 具有活化JNK 和 nuclear factor κB (NFκB) 傳導路徑的功能，其中ZAK 是藉由雙磷酸激酶JNKK2/MKK7 來活化JNK 的路徑。為了驗證ZAK 在細胞的受器者及研究其所引發的訊息傳導的梯瀑效應，J.J.YANG 的研究室以yeast two-hybrid system 從人類的cDNA 基因庫，比對出了ZAK 的結合蛋白(associated proteins；effector)其中之一就是Rho GDP dissociationinhibitor beta (RhoGDIβ)。一般對RhoGDIβ的了解，僅限於其對RhoGTPases的分解作用；然而，究竟RhoGDIβ 的功能，是否真為RhoGTPase 的調控者？本實驗室目前的研究結果，提供了ZAK 的功能是為 RhoGDIβ 激酶的證據。同時在in vitro 的實驗顯示：ZAK 磷酸化RhoGDIβ 後與RhoGDIβ 間呈現物理性的鍵結關係，同時負向調控了RhoGDIβ 所引發的肥大性生長。

因此，假設了ZAK-RhoGDIβ 間這樣的的負向交互調控關係，可以使ZAK 保持在低磷酸化而且失活狀態。

Kinase assay 測得ZAK 的自我磷酸化，結果顯示RhoGDIβ 可同時被ZAK 磷酸化並與ZAK 鍵結，故推論RhoGDIβ 的功能受到ZAK 的調控。在心肌細胞的培養實驗，以Flow cytometry 分析RhoGDIβ-expressing cells可發現當RhoGDIβ 過度表達時，會抑制細胞周期的進行，並引起細胞的肥大性生長。利用SiRNA knockdown RhoGDIβ 表達時，會阻斷細胞的肥大性生長。同時在in vitro 的研究顯示，被ZAK 磷酸化後的RhoGDIβ，會與ZAK 間呈現物理性的交互作用，而RhoGDIβ 的磷酸化又會負向的調控RhoGDIβ 本身的功能。再者，ZAK-RhoGDIβ 間的交互作用，可以使ZAK 保持在低磷酸化的失活狀態。

這兩種蛋白彼此間呈現互相負調控的功能，所以ZAK 可藉由磷酸化來壓抑RhoGDIβ 的功能，而RhoGDIβ 則因此與ZAK 呈現物理性的鍵結。因此利用對ZAK specific 的RNA 干擾方法，在可ZAK- RhoGDIβ expressing cell 的細胞中，knockdown ZAK 的表達後，RhoGDIβ 的功能即可完全恢復。

研究結果顯示RhoGDIβ所引發的H9c2細胞肥大性生長，受到ZAK 的負向調控。在先前研究RhoGDIβ與Rac1 的調控關係的實驗結果顯示，RhoGDIβ藉由控制Rac1 傳譯表達調控細胞肥大性生長及細胞移動，但RhoGDIβ對Rac1 所扮演的調控角色仍然未明。當以siRNA knockdown RhoGDIβ的表達時，會抑制由RhoGDIβ-induced Rac1 的表達與細胞移動現象。

而且，當ZAK 和RhoGDIβ同時表達時，會抑制ZAK 所調控ANF 的表達 。以ZAK specific siRNA knockdown ZAK 的表達，可恢復 RhoGDIβ的活性。RhoGDIβ在H9c2 心肌細胞藉由調控Rac1 表達，來引發細胞的肥大性生長；而且RhoGDIβ調控的細胞移動呈現Rac1 依賴。以siRNA knockdown RhoGDIβ的過度表達時，可降低H9c2 心肌細胞的移動。然而RhoGDIβ所引發H9c2 心肌細胞的移動受到ZAK的負向調控，與細胞的增殖(cell proliferation)並無關聯。

ZAK belongs to the mixed lineage kinases, a family of serine/threonine kinases that are classified as MAP3Ks and can activate the JNK and nuclear factor κB (NFκB) pathway ZAK induces JNK activation through a dual phosphorylation kinase, JNKK2/MKK7.
To identify effectors of ZAK and to study the ZAK signaling cascade,we used a yeast two-hybrid system to isolate ZAK effectors from a human heart cDNA library. One of the isolated cDNAs encoded Rho GDP dissociation inhibitor beta (RhoGDIβ).However, only their ability to sequester RhoGTPases is well understood. Therefore, the question
remains as to whether RhoGDIβ is actually a RhoGTPase regulator.
Overexpression of RhoGDIβ, a Rho GDP dissociation inhibitor, induced hypertrophic growth and suppressed cell cycle progression in a cultured cardiomyoblast cell line. Knockdown of RhoGDIβ expression by RNA interference blocked hypertrophic growth. The further studies demonstrated that RhoGDIβ physically interacts with ZAK and is phosphorylated by ZAK in vitro, and this phosphorylation negatively regulates RhoGDIβ functions.
Moreover, the ZAK-RhoGDIβ interaction may maintain ZAK in an inactive hypophosphorylated form. These two proteins could negatively regulate one another such that ZAK suppresses RhoGDIβ functions through phosphorylation and RhoGDIβ counteracts the effects of ZAK by physical
interaction.
Knockdown of ZAK expression in ZAK- and RhoGDIβ-expressing cells by ZAK-specific RNA interference restored the full functions of RhoGDIβ. At our previous study demonstrated that RhoGDIβ plays a undefined role in regulating Rac1 expression through transcription to induce hypertrophic growth and cell migration and that these functions are
blocked by the expression of a dominant-negative form of Rac1.Knockdown
of RhoGDIβ expression by RNA interference blocked RhoGDIβ-induced Rac1 expression and cell migration.
We demonstrated that the co-expression of ZAK and RhoGDIβ in cells resulted in an inhibition in the activity of ZAK to induce ANF expression. Knockdown of ZAK expression in ZAK-RhoGDI β-expressing cells by ZAK-specific RNA interference restored the activities of RhoGDIβ.

Expression of RhoGDIβ induces hypertrophic growth via
modulation of Rac1 expression in H9c2 cardiac cells. H9c2 cell migration promoted by RhoGDIβ is Rac1 dependent . Knockdown of overexpressed RhoGDIβby siRNA reduces H9c2 cell migration. RhoGDIβ-induced cell migration does not correlate with cell proliferation ，and RhoGDIβ
-induced wound healing is negatively regulated by ZAK.

目錄

中文摘要 II
Abstract: V
序論與文獻回顧 - 1 -
MAPKs的傳導路徑 - 1 -
The GTPase superfamily : regulatory GTPase - 4 -
ZAK蛋白激酶 - 7 -
RhoGDIβ - 9 -
研究動機 - 12 -
實驗材料 - 14 -
實驗方法 - 27 -
ZAK激酶的免疫沉澱試驗與活性測試 - 28 -
Pull-down assay - 30 -
SiRNA knockdown - 31 -
細胞染色及生長評估 - 33 -
北方點墨法 - 34 -
癒合試驗 - 35 -
細胞膜與細胞質的萃取 - 36 -
實驗結果 - 37 -
ZAK與 RhoGDIβ間的交互作用 - 38 -
RhoGDIβ引發H9c2心臟細胞的肥大性生長 - 40 -
在表達RhoGDIβ的H9c2 cells，剔除RhoGDIβ可恢復細胞型態 - 41 -
RhoGDIβ對細胞周期調控蛋白表達的控制效應 - 43 -
RhoGDIβ引發的肥大性生長受到ZAK的調控 - 45 -
RhoGDIβ and ZAK過度表達可逆轉RhoGDIβ調控的細胞周期停滯 - 46 -
RhoGDIβ在H9c2心肌細胞的表達是藉由調控Rac1引發肥大性生長 - 48 -
RhoGDIβ所引發的H9c2細胞移動為 Rac1-dependent - 51 -
以siRNA knockdown RhoGDIβ的過度表達可以減緩H9c2 cell的細胞移動 - 53 -
討論 - 57 -
預期貢獻 - 61 -
參考文獻 - 62 -
圖表與說明： - 71 -
圖一 ZAK引發自我磷酸化及GST-RhoGDIβ的磷酸化 - 72 -
圖二：在 H9c2細胞，RhoGDIβ的表達會阻礙細胞周期的進行 - 73 -
圖三 ： RhoGDIβ引發H9c2心臟細胞的肥大性生長 - 74 -
圖四：以SiRNA knockdown RhoGDIβ 會抑制RhoGDIβ-expressing cells的 移動 - 75 -
圖五(A，B)：RhoGDIβ對Rac1表達的影響 - 76 -
圖五（C，D）：RhoGDIβ對RhoGTPases 的細胞定位 - 77 -
圖六：(A) RhoGDIβ所引發的細胞肥大性生長為 Rac1-dependent - 78 -
圖六：(B) RhoGDIβ所引發的細胞移動為 Rac1-dependent， - 79 -
圖六：(C) RhoGDIβ對細胞周期進行的影響，則非 Rac1-dependent - 80 -
圖七：H9c2 cells各種不同表達的生長曲線 - 81 -
圖八：cyclin-dependent kinase inhibitors p21，p27的表達 - 82 -
圖九(A)：ZAK 可反轉RhoGDIβ 所引發H9c2 細胞的移動 - 83 -
圖九(B)：ZAK 可反轉RhoGDIβ 所引發H9c2 細胞的移動 - 84 -
圖拾：H9c2 和H9c2 細胞表達各式蛋白質的共軛焦顯微鏡的觀察 - 85 -
圖拾一：ZAK反轉RhoGDIβ在H9c2 cells所引發的各種表達 - 86 -
圖十二(A，B)：ZAK 呈現專一性地降低Rac1蛋白與細胞膜的接合量，來負向調控RhoGDIβ的活性 - 88 -
圖十二(C，D)：ZAK 呈現專一性地降低Rac1蛋白與細胞膜接合的量，來負向調控RhoGDIβ的活性 - 90 -
附圖目次
附圖一：各種ZAK –RhoGDIβ的鍵結關係與被ZAK 的磷酸化狀( A ) RhoGDIβ與各種ZAK鍵結親和力 - 91 -
( B) RhoGDIβ與各種ZAK的鍵結 - 93 -
附圖二： RhoGDIβ引發H9c2心臟細胞的肥大性生長 - 94 -
( A) ：rhodamine-conjugated phalloidin免疫染色： - 95 -
( B ) ：H9c2 cells與轉殖pTet-RhoGDIβ的相對細胞型態大小 - 96 -
附圖三( A )：H9c2 cells的生長速率 - 97 -
( B )：在RhoGDIβ表達細胞中，細胞周期調控蛋白的表達與活性 - 98 -
附圖四：( A ) ZAK反轉RhoGDIβ在H9c2 cells所引發的細胞肥大性增殖 - 99 -
( B )：ZAK反轉RhoGDIβ在H9c2 cells所抑制的生長速率 - 100 -
附圖五：RhoGDIβ 鍵合於ZAK 的encompassing residues 600–700的區域 - 101 -
(附圖 六)：ZAK接合位的胺基酸片段(ZAK 6–7)可被GST-RhoGDIβ - 102 -
(附圖 七)The switch function of the regulatory GTPase - 103 -
(附圖 八) Signal Transmission via Ras Proteins - 104 -
(附圖九)MAPKs: function, regulation, role in cancer and therapeutic targeting - 105 -

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