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  • 學位論文

細胞週期蛋白Cyclin E和Cdk2複合體調控 多種轉錄因子STAT蛋白之活性研究

Cyclin E-Cdk2 Complex Regulate the Expression Level and Transcriptional Activity of STAT Proteins

指導教授 : 黃銓珍

摘要


Janus kinase (JAK) / signal transducer and activator of transcription (STAT)途徑,是一條極為重要、從膜到核的信號轉導系統。然而它所影響範疇包括了細胞增生、細胞命運的決定、組織的發育以及免疫系統反應。哺乳類動物中,JAK家族有JAK1, JAK2, JAK3 和TYK2,STAT家族則有STAT1-6。STAT會受到JAK的磷酸化而活化,進入細胞核內調控其目標基因的表現。 本實驗室先前已完成五種圓斑河豚信號傳遞轉錄因子STATs (STAT1, STAT3-STAT6)之基因選殖,也構築含JAK1之kinase domain(JH1)之重組STATs,有STAT1-JH1、STAT3-JH1、STAT4-JH1、STAT5-JH1和STAT6-JH1五種。爲進一步了解他們的蛋白質特性,分別將五種基因轉染到COS-1細胞,由西方墨點法分析,STAT-JH1融合蛋白能夠自行進行酪胺酸磷酸化作用;利用免疫螢光染色發現,STAT-JH1在細胞質和細胞核都可以見到有所表現,不同於STATs只在細胞質表現。STAT-HA-JH1可活化β-casein基因啟動子之轉錄活性,也會去負向調控14-3-3 η(eta)啟動子的轉錄活性,代表了STAT-JH1在無外界刺激下,可持續處於活化狀態,並且具有轉錄活性。 而本論文進一步作細胞週期蛋白Cyclin E-Cdk2複合體調控STAT轉錄因子之蛋白質表現與轉錄活性之研究。將STAT和Cyclin E、Cdk2共轉染後,發現STATs(活化或未活化)蛋白表現量有顯著的降低;而用同樣的方式,再外加上蛋白酶抑制劑,就可以使STATs蛋白質原本的低表現量有所回升,顯示蛋白降解的現象可能是透過ubiquitin-proteasome程序。從免疫沉澱實驗中證明了Cdk2會和STATs有蛋白質與蛋白質之間的交互作用,然而在STAT和Cyclin E、Cdk2共轉染後,STATs對β-casein啟動子之轉錄活性明顯被抑制。這些結果代表了Cyclin E-Cdk2除了調節細胞週期G1-S時期外,同時也會參與JAK-STAT訊息傳遞。

並列摘要


In mammals, the JAK-STAT pathways are known to play very important roles in cytokine signal transductaion. Members in this pathway include the Janus kinases (JAKs) family containing JAK1, JAK2, JAK3 and TYK2 and the STATs (signal transducer and activator of transcription), which contain STAT1 to STAT6. The activation of STATs provides a rapid method for transducing signal from membrane into nucleus, and this mechanism is conserved from insects to human. However, the functional mechanism of this pathway is pooly understood in fish. We have constructed five STAT genes from pufferfish and also prepared STAT-JH1 chimeric proteins generated by fusion of the kinase domain (JH1 domain) of JAK1 kinase to the C-terminal end of STAT. To examine the function of STAT-JH1, we performed transfection study in COS-1 cells. Immunoblotting of cell lysate showed that the product of STAT-JH1 could undergo auto-tyrosine-phosphorylation. By immunocytochemistry, STAT-JH1 proteins are not only localized in cytoplasm but also could translocated into nucleus. In luciferase assay experiment, STAT-JH1 could enhance the transcriptional activity of β-casein promoter and inhibit the transcriptional activity of 14-3-3 ηpromoter. This finding indicates that STAT-JH1 proteins have transactivation ability without any extracellular stimulus. Moreover, we studied the regulation of Cyclin E-Cdk2 on the protein expression and transcriptional activity of pufferfish STAT proteins. Different STATs were cotransfected with either Cyclin E or Cdk2 or Cyclin E and Cdk2 into COS-1 cells and the expression level of STAT proteins were analyzed. The results show that Cyclin E-Cdk2 could reduce STAT proteins expressions (STAT-HA or STAT-HA-JH1). In the presence of proteasome inhibitor,the low STAT proteins level will be rescued. By immunoprecipitation, STAT-HA-JH1 fusion protein can associate with Cdk2. We also found the transcriptional activity ofβ-casein promoter regulated by STAT-HA-JH1 was obviously inhibited by co-expression Cdk2 and Cyclin E. In summary, these data suggest that Cyclin E-Cdk2 is not merely one of the key regulators of the G1/S transition in the cell cycle. They maybe have connection with JAK/STAT pathway.

並列關鍵字

STAT Cyclin E Cdk2

參考文獻


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