Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/22987
標題: Cdk5蛋白對於MCF-7乳癌細胞中p21蛋白之分布的探討
Investigation of Cdk5-dependent p21 Localization in MCF-7 Breast Cancer Cell
作者: 游舜期
You, Shuen-Chi
關鍵字: Cdk5
Cdk5
p21
cell cycle
p21
細胞週期
出版社: 生命科學系所
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摘要: 細胞週期主要分為四個時期,分別為G1、S、G2與M (mitosis) 期,每一時期由不同的Cdk與cyclin調控。在哺乳動物細胞中,Cdk家族主要參與細胞週期的是Cdk1 (Cdc2)、Cdk2、Cdk4和Cdk6。Cdk5因其序列與Cdk1和Cdk2具有高度相似性,且在催化區 (catalytic subunit) 區域與Cdk家族一起被歸類於CMGC (containing CDK, MAPK, GSK3, CLK families) kinase中,因此將Cdk5歸於Cdk家族中的一員,然而Cdk5對於細胞週期的調控到目前為止仍不明確,且在部分研究指出Cdk5對於細胞週期是不具有調控的機制。細胞週期除了正向調控的Cdk與cyclin外,亦有我們所熟知的負向調控因子cyclin-dependent kinase inhibitor (CKI),根據CKI的初級胺基酸結構與抑制特定Cdk的專一性可將之歸為兩大類,分別是INK4 (Inhibitors of Cdk4) 與Cip/Kip (Cdk interacting protein/Kinase inhibitory protein) 家族。Cip/Kip家族中的p21,其負向影響細胞週期主要藉由影響Cdk和cyclin E、D和A的鍵結與抑制Cdk活性。近年來,多篇研究指出p21經過不同激酶的磷酸化轉錄後修飾,不僅影響p21本身的穩定度,亦促使p21正向調控細胞週期的功能。本研究目的在於探討Cdk5是否可能經由影響p21而調控細胞生長。結果顯示乳癌細胞MCF-7中內生性的Cdk5與p21之間有生化交互作用。在細胞內蛋白核質分離實驗,顯示內生性p21會受到Cdk5抑制劑處理而增加在細胞核的分布,並在過度表現外源Cdk5或p35而減少在細胞核的分布。進一步利用表現外源GFP-p21融合蛋白,以免疫螢光方式觀測在過度表現外源Cdk5或p35蛋白下GFP-p21在胞器內的分布,結果顯示Cdk5或p35蛋白都會使GFP-p21傾向座落在細胞質,因此推測Cdk5活性對於p21在細胞內分布調控佔有相當的角色。此外,為了研究Cdk5對於細胞週期是否具有調控功能,藉由建立Cdk5穩定細胞株並檢測其細胞週期,觀察到在64小時內的不同時間點血清再處理 (serum add-back),相較於控制組細胞株,Cdk5穩定細胞株的細胞週期運行有較快的情形。在多篇文獻指出p21座落在細胞質對於細胞週期有正向調控功能,且本實驗觀察到過度表現Cdk5可促使p21分布在細胞質,加上Cdk5穩定細胞株實驗中發現Cdk5對於細胞週期有促進的效果,因此Cdk5是否藉由影響p21在胞器的分布來達到間接調控細胞週期需要更進一步探討。而這些訊息調控的基礎研究,冀望能提供基礎及臨床醫學新的致癌機轉,並在未來能開發相關藥物應用於臨床治療。
Cell cycle control is primarily regulated by the members of cyclin-dependent kinase family with their specific cyclins. Cyclin-dependent kinase 5 (Cdk5) was identified as a member of Cdk family because of the sequence homology. However, the activator of Cdk5 is not the traditional cyclins but the p35 or p39. In addition, the role of Cdk5 in cell cycle regulation is currently ambiguous. The sequential Cdks activation and inactivation control the progression of entire cell cycle. The cyclin-dependent kinase inhibitors (CKIs), classified into the CIP/KIP and INK4 family, provide one of the mechanisms for Cdks inactivation. p21, a member of CIP/KIP family, inactivates Cdks by interfering the protein interaction between Cdks and cyclins. Besides, the interaction of Cdk5 and p21 was also reported. Interestingly, recent literatures indicated that cytoplasmic p21 could positively regulate cell cycle and play anti-apoptotic function in opposition to its nuclear functions. Therefore, our purpose is to investigate if Cdk5 could regulate the p21 localization and its biological function in cell cycle. At first, the endogenous protein interaction in MCF-7 between Cdk5 and p21 was identified by immunoprecipitation. The nuclear p21 was increased by the dose-dependent treatments of Cdk5 inhibitor (roscovitine) and decreased by overexpression of Cdk5 or p35. Furthermore, we found that p21-GFP fusion protein was accumulated in cytoplasm after overexpression of Cdk5 or p35 with serum add-back. According to these results, Cdk5 might play a vital role in regulating the cytoplasmic and nuclear distribution of p21. In order to determine the role of Cdk5/p21 in cell cycle regulation, MCF-7 stable cell lines were established. We found that stable overexpression of Cdk5 promoted the cell cycle process when cells reenter cell cycle after serum add-back. Base on current results, we hypothesize that subcellular localization of p21 protein might be regulated by Cdk5 and the cell cycle process was therefore promoted.
URI: http://hdl.handle.net/11455/22987
其他識別: U0005-3006200902420300
文章連結: http://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-3006200902420300
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