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http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/48241
標題: | 探討SIK2蛋白於調控LDCVs分泌之功能角色 The functional role of SIK2 in regulating large dense core vesicles (LDCVs) secretion |
作者: | Chung-Ju Kao 高仲儒 |
指導教授: | 周涵怡(Han-Yi Chou) |
關鍵字: | 分泌,SIK2,PKA,β細胞,胰島素小泡, secretion,SIK2,PKA,β cells,insulin vesicles, |
出版年 : | 2011 |
學位: | 碩士 |
摘要: | 唾液腺是人體口腔中非常重要的腺體,它的分泌提供了保護功能以及消化功能。從免疫組織染色中我們發現屬於AMPK家族一員的SIK2蛋白在唾液腺中有高度的表現。SIK2的Serine587是PKA潛在的磷酸化位置,而PKA是腺體分泌中最主要的調控因子。我們發現SIK2-S587周圍的序列在脊椎動物中是高度保存的,同時我們證明了PKA與SIK2存在於同一個複合體,而且PKA可以在in vivo和in vitro的實驗中磷酸化SIK2-S587。另外我們也發現SIK2-S587的磷酸化確實會發生於唾液腺以及其他以Large dense core vesicle (LDCV)進行分泌的腺體。利用以LDCV分泌胰島素的β細胞中,我們發現葡萄糖的刺激可以快速的促使SIK2-S587的磷酸化,而且這個磷酸化的現象和PKA的活化有關。免疫螢光染色也顯示SIK2-pS587與PKA同時存在於部份的胰島素小泡中,暗示著SIK2和PKA的交互作用可能和胰島素小泡的調控相關。另外,經由TIRF顯微鏡我們也發現,在葡萄糖刺激後,SIK2-pS587和細胞膜周圍胰島素小泡的交互作用明顯上升,顯示SIK2-S587的磷酸化可能參與在β細胞中控制胰島素小泡運輸的機制上。這些證據暗示了SIK2可能在調控LDCV的分泌中扮演一個重要的角色。 Salivary glands are an important secretory tissue of the oral cavity. From Immunohistochemical staining, we found that SIK2, a member of the AMPK family proteins, is highly expressed in salivary glands. The Ser587 residue of SIK2 has been identified as a putative phosphorylation site of PKA, which is a principal modulator in the regulation of secretion. Here we demonstrate that the context sequence around Ser587 residue of SIK2 is highly conserved among veterbrates, and identify PKAβ as the kinase that physically associates and phosphorylates SIK2 at S587 in vivo and in vitro. This phosphorylation occurs not only in the salivary glands but also in various Large dense core vesicle (LDCV)-secreting glands. Using the LDCV-mediated secretion of insulin model of β islet cells, we found that SIK2-S587 is rapidly phosphorylated upon glucose stimulation, and that this phosphorylation strongly correlates with PKA activation under physiological conditions. In Rinm5F insulinoma cells, SIK2-pS587 and PKAβ colocalize to insulin-containing LDCVs, while TEM observations indicate that SIK2-S587 phosphorylation can occurs as early as in reserve pool insulin vesicles. Our kinetics studies indicate that massive translocation of SIK2-pS587 containing insulin vesicles occurs upon glucose stimulation as observed under Total Internal Reflection Fluorescence Microscopy (TIRF). Taken together, our data suggest an important role for SIK2 in LDCV secretion, and that SIK2 may crosstalk with PKA to regulate insulin vesicle translocation in β islet cells. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/48241 |
全文授權: | 有償授權 |
顯示於系所單位: | 口腔生物科學研究所 |
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