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研究生:連莉莉
研究生(外文):Lily Lien
論文名稱:探討CaMKKβ與14-3-3之結合
論文名稱(外文):The interaction of CaMKKβ and 14-3-3
指導教授:林棋財
指導教授(外文):Chi-Tsai Lin
學位類別:碩士
校院名稱:國立臺灣海洋大學
系所名稱:生物科技研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2005
畢業學年度:93
語文別:中文
論文頁數:75
中文關鍵詞:攜鈣素蛋白激酶激酶14-3-3
外文關鍵詞:CaMKK14-3-3
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本論文主要探討 CaMKKβ與其結合蛋白 14-3-3之交互作用,以及此交互作用對 CaMKKβ生理活性以及功能的影響。在細胞中,calcium/calmodulin (CaM) kinase cascade是傳遞鈣離子訊號的主要路徑之一,其包含有 CaMKK及其下游受質 CaMKI以及 CaMKIV等分子。在哺乳類中,CaMKK為橫向同源 (Paralog),具有 CaMKKα以及 CaMKKβ兩種不同的基因。之前我們實驗室利用 cDNA library screen配合 RT-PCR方法選殖出兩種亞型的人類 CaMKKβ cDNA,其長度分別為 5.4 kb (CaMKKβ1) 以及 2.5 kb (CaMKKβ2),兩者皆表現於所有組織中,而以 β1亞型為主要的表現型。為了進一步瞭解 CaMKKβ的功能及其調控機制,我們藉由酵母菌雜交技術選殖其結合蛋白,發現 14-3-3γ可以和 CaMKKβ結合,進一步分析 CaMKKβ的氨基酸序列,也發現其 C端含有 14-3-3蛋白可辨識的區塊 (motif)(508RSLSAP513)。本論文之主要目的,即是進一步探討 14-3-3與 CaMKKβ的結合機制,以及其對 CaMKKβ功能之調控。在此,我們利用細胞內免疫沈澱以及細胞外 GST融合蛋白抓取實驗證實 14-3-3γ可以和 CaMKKβ結合,並進一步建立不含 C端或含單點突變 (S511A) 之突變型 CaMKKβ確定 508RSLSAP513為其與 14-3-3γ結合的主要區塊。文獻指出,Protein kinase A (PKA) 的活化會抑制 CaMKKα的激酶活性,我們也發現利用Forskolin活化 PKA,除了會降低 CaMKKβ的激酶活性之外,並會增強 CaMKKβ與 14-3-3γ的結合。為了分析 PKA磷酸化 CaMKKβ對其與 14-3-3交互作用的影響,我們進一步利用表現不同片段以及單點突變的 CaMKKβ對其蛋白序列上四個典型可以被 PKA辨識並磷酸化 (R/K-R/K-X-S/T) 的區塊 (97RKLS100、142RRPT145、492RKRS495、519KKPT522) 進行分析,發現 PKA能透過磷酸化 492RKRS495以及519KKPT522,增強 CaMKKβ與 14-3-3γ的結合。此外,我們也利用免疫螢光染色以及共軛焦顯微鏡來探測 CaMKKβ在細胞內的分佈,發現含有 S511單點突變以及去除 C端 (Δ473-588 a.a.) 之突變型 CaMKKβ皆較常分布於細胞核中,推測能利於其作用於下游之 CaMKIV,並促進其轉錄功能。綜合以上實驗結果,顯示 14-3-3γ與CaMKKβ的結合具有負向修飾 CaM kinase cascade的能力。
The purposes of this thesis are to confirm the interaction of CaMKKβ and 14-3-3 and to characterize the functional consequence of this interaction on CaMKK-mediated signaling pathway. The CaM kinase cascade is composed of CaMKK and its downstream substrates CaMKI and CaMKIV. CaMKKα and CaMKKβ are paralogs in mammalian cells. Based on cDNA library screening in conjunction with PCR cloning methods, we have previously isolated two human CaMKKβ cDNA species, CaMKKβ1 and CaMKKβ2; both are ubiquitously expressed with CaMKKβ1 as the predominant form in brain, the site of highest expression. To explore the biological function of human CaMKKβ, we have employed yeast-two hybrid system to isolate its interacting proteins, and identified 14-3-3γ as one of the proteins that interact with CaMKKβ. The aim of this study is to confirm and characterize the interaction between CaMKKβ and its interacting protein 14-3-3γ. By in vivo co-immunoprecipitation and in vitro GST pull down assays, we confirmed that 14-3-3γ binds to CaMKKβ. In addition, we have also mapped the 14-3-3 interaction site located at 508RSLSAP513, and shown that mutation of S511 abolished this interaction. PKA is known to suppress the kinase activity of CaMKKα. In our study, we also found that forskolin treatment does not only suppress the kinase activity of CaMKKβ but also enhance its interaction with 14-3-3γ. To further characterize this effect, we examined the binding of 14-3-3γ to the wild-type CaMKKβ and its mutants on which one or more of the four consensus PKA phosphorylation sites (97RKLS100, 142RRPT145, 492RKRS495, and 519KKPT522) are removed or mutated. We showed that PKA enhances 14-3-3γ binding to CaMKKβ through phosphorylation of residues S495 and S522 within 492RKRS495, and 519KKPT522 located at the C-terminus of CAMKKβ. In the process of examining whether 14-3-3 binding may affect subcellular localization of CaMKKβ, we found that mutation of S511 or truncation of the C-terminus facilitates the nuclear import of CaMKKβ, suggesting that 14-3-3 may sequester CaMKKβ in cytosol. Together, our data suggest that 14-3-3γ may act as a negative modifier in CaM kinase pathway by interacting with CaMKKβ.
目錄 2
A. 圖目錄 4
B. 縮寫表� � � � � � � � � � � � � � � � � � � 5
C. 中文摘要� � � � � � � � � � � � � � � � � � 6
D. 英文摘要 8
E. 序論
� 一、鈣離子與細胞生理� � � � � � � � � � � � � � 10
� 二、CaM kinase family 11
三、CaMKK � � 13
四、14-3-3 � 15

F. 研究目標 18

G. 材料與方法
� 一、� 酵母菌雜交技術 (yeast two hybrid)� � � � � �� � �� 19
二、細胞培養 (cell culture)� 20
� 三、轉染反應 (transfection) 21
� 四、質體製備 22
� 五、細胞蛋白定量 23
六、免疫沈澱 (immunoprecipitation;IP) 24
七、西方墨點轉漬分析 (western blot) 24
八、GST融合蛋白的製備 (GST-protein preparation) � 26
� 九、GST 融合蛋白抓取實驗 (GST pull-down assay) � 27
� 十、激酶活性測試 (kinase activity assay) � � 27
� 十一、Luciferase reporter assay � 27
� 十二、聚合酶鏈鎖反應鑲嵌定點突變法
� � � (site-directed mutagenesis PCR-chimeric method) � 28
十三、免疫螢光染色 (Immunofluorescence stain) � 29

H. 實驗痕G
一、CaMKKβ與 14-3-3之交互作用 � � � 30
� � 1. 確認 14-3-3為 CaMKKβ之結合蛋白 � � � �� � � � � � 30
� � 2. 確認 14-3-3γ與 CaMKKβ1的結合位置為 S511 � 31
二、14-3-3γ對於 CaMKKβ1激酶活性的影響 � � 32
1. 14-3-3對 CaMKKβ可能扮演負向調控之角色 � � � � � � 32
� � 2. PKA的活化增強 CaMKKβ與 14-3-3γ之結合 � � � � � � � � 33
� � 3. PKA的活化減少 CaMKKβ之激酶活性 � � � � � � � � � � 33
4. 14-3-3對 CaMKKβ具負向修飾控其激酶活性之能力 � � � � 33
5. PKA對 CaMKKβ與 14-3-3γ結合之影響 � � � � 34
6.不同 14-3-3γ結合程度之 CaMKKβ 激酶活性� � � � �� � � � 35
� 三、14-3-3γ對 CaMKKβ在細胞中分佈的影響 36

I. 討論 � � � � � � � � � � � � � � 38
J. 參考文獻 43
K. 附圖� � � � � � � � � � � � � � � � � � � 51
L. 附錄� 74
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