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標題: | 鑑定第二型水通道蛋白絲胺酸269之激酶 Identification of protein kinases responsible for aquaporin-2 phosphorylation at serine 269 |
作者: | Tzu-Hsien Chan 詹子嫻 |
指導教授: | 余明俊 |
關鍵字: | 第二型水通道蛋白,抗利尿激素,激?,磷酸化,水分再吸收, aquaporin-2,vasopressin,kinases,phosphorylation,water reabsorption, |
出版年 : | 2019 |
學位: | 碩士 |
摘要: | 腎臟的水分再吸收作用發生於腎臟集尿管,並且受到抗利尿激素(vasopressin)的調控。在vasopressin的刺激下,集尿管細胞內的環磷酸腺苷(cAMP)濃度提升,促使第二型水通道蛋白(aquaporin-2,簡稱AQP2)的絲氨酸269(S269)磷酸化,使得AQP2從集尿管細胞的胞內體被轉運到尖頂膜(apical membrane),增加AQP2在apical membrane的數量,以促進水分的再吸收。在先前的研究中指出,S269磷酸化可以延長AQP2停留在apical membrane的時間,進而增加細胞對水分的通透性,以利水分再吸收的進行。然而,目前對於磷酸化S269的激酶(kinase)並不清楚。因此,我們利用小鼠腎臟集尿管細胞(mpkCCD)建立一個找尋S269激酶的系統。mpkCCD常用於AQP2磷酸化以及胞內運輸的研究,它需要耗費約三至四天的時間進行極化後,再以vasopressin刺激四天誘導內生性的AQP2生成,才能進行AQP2的相關研究。但是,小鼠的激酶總共有521個,若以極化系統來進行研究,勢必耗費時日。在此,我們改以沒有極化的mpkCCD作為研究S269激酶的系統,來因應極化系統耗時的問題。我們先在沒有極化的mpkCCD表達外源性的AQP2,以解決沒有內生性AQP2的問題。再以毛喉素(forskolin)模擬在vasopressin刺激下,胞內cAMP濃度提升的情形。我們發現forskolin可以顯著提升未極化細胞中S269的磷酸化,這樣的結果說明著,受到cAMP活化的激酶,可能參與S269磷酸化。我們也發現,在forskolin的刺激下,AQP2會從胞內被運送至membrane上。上述所發現的結果皆與極化系統的現象一致,代表著未極化的mpkCCD中,同樣具有激酶、磷酸酶(phosphatase),和參與運輸蛋白的分子,以完成AQP2的磷酸化和轉運。接著我們以貝氏定理對521個激酶進行排序,有七個激酶分布於前四名。其中一個激酶是protein kinase A (PKA),可以促使S269磷酸化。抑制PKA基因轉譯或是抑制PKA活性,皆導致S269磷酸化降低。而在先前的研究已經發現,S269磷酸化需要PKA,但PKA並非是直接磷酸化S269的激酶。縱使我們並未發現磷酸化S269的激酶,但也證明了我們發展出來的系統的確可以取代極化系統來進行AQP2的相關研究。 Aquaporin-2 (AQP2) is a water channel protein regulated by vasopressin, which triggers AQP2 trafficking to the apical plasma membrane of the kidney collecting duct principal cells for water reabsorption. Vasopressin elevates intracellular cyclic AMP (cAMP), resulting in AQP2 phosphorylation at serine 269 (S269) and trafficking to the apical plasma membrane. It has been shown that S269 phosphorylation is associated with apical AQP2 localization most likely by promoting apical AQP2 retention; however, the kinase responsible for S269 phosphorylation is still unknown. Here, we used non-polarized mpkCCD cells to establish a kinase screening system. The mouse mpkCCD cells have been an instrumental collecting duct cell model for studying AQP2 phosphorylation and trafficking, but the cells require a time-consuming polarization process before they can respond to vasopressin. We sought to circumvent this issue by examining AQP2 phosphorylation and trafficking in non-polarized AQP2-overexpressing mpkCCD cells treated with forskolin, which elevates intracellular cAMP. We found that forskolin significantly induced S269 phosphorylation as observed in polarized mpkCCD cells treated with vasopressin. Thus, AQP2 S269 phosphorylation likely involves kinases that are activated by cAMP. In response to forskolin, AQP2 was found in the plasma membrane and was phosphorylated at S269, similar to those observed in the polarized cells. Thus, the non-polarized cells are equipped with kinases, phosphatases, and trafficking molecules for AQP2 phosphorylation and trafficking as the polarized cells. Using Bayes' theorem to rank all 521 mouse kinase, top four kinase groups constituted with 7 kinases stood out from the rest of kinases. One of the top-ranked kinases is protein kinase A (PKA), which induces S269 phosphorylation. We found that PKA knockdown reduced S269 phosphorylation and membrane localization treated with forskolin. PKA Inhibitory assay showed result consistent with the PKA knockdown result. Previous study has shown that PKA acts indirectly on S269 phosphorylation. Thus, we conclude that non-polarized AQP2-overexpressing mpkCCD cells can be substituted for polarized mpkCCD cells for kinase screening. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/73767 |
DOI: | 10.6342/NTU201903856 |
全文授權: | 有償授權 |
顯示於系所單位: | 生物化學暨分子生物學科研究所 |
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