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標題: | 探討 CDK9 調控核受器 LRH-1 的分子機制 The molecular mechanism of CDK9 in regulation of nuclear receptor LRH-1 |
作者: | Yu-Fang Wang 王裕方 |
指導教授: | 胡孟君(Meng-Chun Hu) |
關鍵字: | 核受器,磷酸化, LRH-1,CDK9, |
出版年 : | 2015 |
學位: | 碩士 |
摘要: | Liver receptor homolog-1 (LRH-1;NR5A2) 隸屬於孤兒核受器,對於發育、膽固醇運輸、膽酸平衡以及固醇類荷爾蒙生成相當重要。然而,目前對於調控LRH-1的相關機制所知仍有限。本實驗室利用酵母菌雙雜交技術,於老鼠肝臟 cDNA library中找到激酶 CDK9 與LRH-1會有相互作用。經由共同免疫沉澱法,證實CDK9與LRH-1在哺乳細胞中有交互作用;利用免疫螢光染色法,偵測到CDK9 同時存在於細胞核與細胞質,在細胞核中的分布與 LRH-1有重疊。利用In vitro kinase assay 證實LRH-1可直接受到CDK9的磷酸化修飾。當同時轉染CDK9與LRH-1於HEK293T細胞中,發現 LRH-1蛋白質的量會降低;若將CDK9過量表現於HepG2肝癌細胞中,會造成內源性LRH-1蛋白質量減少,但不會影響LRH-1 mRNA表現量。另外,加入CDK9抑制劑 DRB 或以 shRNA減低CDK9在HepG2細胞的表現量,會使LRH-1蛋白質量上升,說明了CDK9會調控LRH-1蛋白質的量。利用蛋白質衰減測定,發現CDK9可降低LRH-1蛋白質半衰期。此外,CDK9調控LRH-1蛋白質量的能力會受到蛋白酶體抑制劑MG-132所抑制,且CDK9會增加LRH-1與聚泛素鏈結合。這些結果顯示,CDK9會促進LRH-1蛋白質經由蛋白酶體路徑而降解。
利用啟動子活性分析,發現CDK9 會抑制LRH-1調控 Small heterodimer partner (SHP) 與Glucokinase (GCK) 啟動子的轉錄活性。SHP與GCK參與肝細胞重要的代謝反應,增加或減少CDK9的量,會影響SHP與GCK在HepG2的表現。此外,給予CDK9抑制劑會增加 Insulin 訊息傳遞路徑中磷酸化 pAKT 與 pErk1/2蛋白質量。綜合結果說明,CDK9對於調控LRH-1蛋白質,以及肝細胞代謝相關反應,扮演著重要的角色。 The orphan nuclear receptor liver receptor homolog-1 (LRH-1; NR5A2) plays a critical role in development, reverse cholesterol transport, bile-acid homeostasis and steroidogenesis. However, little is known about the regulation of LRH-1 protein. We previously applied yeast two-hybrid approach to search for LRH-1 interacting proteins in a mouse liver cDNA library and a kinase CDK9 was identified as a novel interaction protein of LRH-1. Immunoprecipitation experiments confirmed the interaction between CDK9 and LRH-1 in mammalian cells. The immunofluorence assay revealed that CDK9 present in nucleus and cytoplasm and it colocalized with LRH-1 in the nucleus. In vitro kinase assay showed that LRH-1 can be phosphorylated by CDK9. We found that co-expression of CDK9 significantly decreased the protein levels of LRH-1 in HEK293T cells. Overexpression of CDK9 in hepatoma HepG2 cells decreased the protein levels of endogenous LRH-1, whereas it had no effects on LRH-1 mRNA levels. In addition, pharmacological inhibition of CDK9 kinase activity or CDK9 knockdown caused an increase in endogenous LRH-1 protein levels in HepG2. These demonstrate that CDK9 can regulate LRH-1 protein levels. By protein turnover assay, we found that CDK9 decreased LRH-1 protein half-life. In addition, CDK9-mediated LRH-1 protein levels were inhibited by the proteasome inhibitor MG-132. Furthermore, CDK9 increased LRH-1 polyubiquitination. These results indicate that CDK9 may promote LRH-1 protein degradation through proteasome pathway. In promoter assays, CDK9 reduced the LRH-1-regulated small heterodimer partner (SHP) and glucokinase (GCK) promoter activity. SHP and GCK involved in the hepatic metabolism. The expression of SHP and GCK was affected by CDK9 overexpression or knockdown. In addition, CDK9 inhibitor upregulated the levels of phosphorylated AKT and Erk1/2 proteins in insulin signaling pathway. Collectively, these results suggest that CDK9 may play an important role in modulating LRH-1 protein and hepatic metabolism. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/55094 |
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