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標題: | 探討Mark2和Arhgap21在Aqp2絲胺酸269磷酸化及頂尖膜運輸的作用 Investigating roles of Mark2 and Arhgap21 in Aqp2 serine 269 phosphorylation and apical trafficking |
作者: | 林怡君 Yi-Jiun Lin |
指導教授: | 余明俊 Ming-Jiun Yu |
關鍵字: | 第二型水通道蛋白,絲胺酸269磷酸化,頂尖膜運輸,抗利尿激素, AQP2,serine 269 phosphorylation,apical trafficking,vasopressin, |
出版年 : | 2024 |
學位: | 碩士 |
摘要: | 第二型水通道蛋白 (AQP2) 是一種受抗利尿激素 (vasopressin) 所調控的水通道蛋白,在腎臟集尿管細胞中負責水分再吸收。受抗利尿激素刺激時,第二型水通道蛋白會從細胞內囊泡運送到細胞頂尖膜,從而進行滲透性水分輸送。第二型水通道蛋白在細胞頂尖膜的定位與其C端PDZ motif中的絲胺酸269磷酸化密切相關。負責第二型水通道蛋白絲胺酸269磷酸化的激酶以及參與頂尖膜運輸的蛋白質目前尚不清楚。我們先前發現,給予抗利尿激素的刺激時,在Vps35表現量降低 (knockdown) 的細胞中,位於回收型囊泡 (Rab11-positive recycling endosomes) 的第二型水通道蛋白發生了絲胺酸269磷酸化。利用TurboID biotin ligase及蛋白質體學之方法,鑑定位於回收型囊泡附近的蛋白質,其中包括Mark2和Arhgap21。我們假設Mark2會磷酸化第二型水通道蛋白的絲胺酸269,而絲胺酸269磷酸化的第二型水通道蛋白結合具有PDZ domain的Arhgap21,這是一種抑制RhoA的GTPase活化蛋白,藉此促進F-actin解聚,使第二型水通道蛋白能插入至頂尖膜。與上述假設一致的是in vitro kinase assay顯示Mark2磷酸化合成的第二型水通道蛋白peptide中的絲胺酸269。然而,在小鼠腎臟皮質集尿管 (mpkCCD) 細胞中,Mark2表現量降低 (knockdown) 以及Mark抑制劑皆未影響抗利尿激素所誘導的絲胺酸269磷酸化。Arhgap21表現量降低 (knockdown) 未影響mpkCCD細胞中F-actin的結構,也未影響第二型水通道蛋白的頂尖膜運輸。為了找到負責磷酸化第二型水通道蛋白絲胺酸269的激酶,我們使用貝氏定理結合各種資料庫,將521種小鼠的激酶進行了排序。Pak2排名第一。Pak2表現量降低 (knockdown) 使mpkCCD細胞無法極化,降低抗利尿激素所誘導的第二型水通道蛋白表達,仍需要進一步探討其功能。 Aquaporin-2 (AQP2) is a vasopressin-regulated water channel protein responsible for water reabsorption in kidney collecting duct cells. In response to vasopressin, AQP2 traffics from intracellular vesicles to the apical plasma membrane, where it mediates osmotic water transport. Apical localization of AQP2 is tightly associated with AQP2 phosphorylation at serine 269 in the COOH-terminal PDZ motif. The kinases and the trafficking machineries responsible for serine 269 phosphorylation and apical AQP2 trafficking remain unknown. We previously found that AQP2 can be phosphorylated at serine 269 in response to vasopressin while in Rab11-positive recycling endosomes in Vps35 knockdown cells. TurboID biotin ligase-based proximity proteomics identified proteins in the vicinity of Rab11-positive recycling endosomes, including microtubule affinity regulating kinase 2 (Mark2) and Rho GTPase-activating protein 21 (Arhgap21). We hypothesized that Mark2 phosphorylates AQP2 at serine 269 and that serine 269 phosphorylated AQP2 recruits PDZ domain-containing Arhgap21, a GTPase-activating protein for RhoA inhibition, to promote F-actin depolymerization for AQP2 insertion at the apical membrane. Consistent with the hypothesis, in vitro kinase assay showed that Mark2 phosphorylated AQP2 serine 269 in a synthetic AQP2 peptide. However, Mark2 knockdown and Mark family kinase inhibitor did not affect vasopressin-induced serine 269 phosphorylation in the mpkCCD cells. Arhgap21 knockdown did not affect F-actin organization and apical AQP2 trafficking in the mpkCCD cells. To identify new protein kinase candidates for AQP2 serine 269 phosphorylation, we used Bayesian analysis to rank all 521 mouse protein kinases based on previously reported datasets. Pak2 was the top candidate. Pak2 knockdown impaired mpkCCD cell polarization and vasopressin-induced AQP2 expression, obliging further investigation for functional implications. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/91670 |
DOI: | 10.6342/NTU202400734 |
全文授權: | 未授權 |
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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