Investigation of Cdk5-dependent p21 Localization in MCF-7 Breast Cancer Cell

• Main Authors: Shuen-Chi You, 游舜期
• Other Authors: Ho Lin
• Format: Others
• Language: zh-TW
• Online Access: http://ndltd.ncl.edu.tw/handle/44070351738309364778

碩士 === 國立中興大學 === 生命科學系所 === 97 === 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.