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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 周涵怡(Han-Yi Chou) | |
| dc.contributor.author | Chung-Ju Kao | en |
| dc.contributor.author | 高仲儒 | zh_TW |
| dc.date.accessioned | 2021-06-15T06:49:55Z | - |
| dc.date.available | 2012-03-03 | |
| dc.date.copyright | 2011-03-03 | |
| dc.date.issued | 2011 | |
| dc.date.submitted | 2011-02-18 | |
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(2011) SIK2 is a key regulator for neuronal survival after ischemia via TORC1-CREB, Neuron 69, 106-119. 48. Burchfield, J. G., Lopez, J. A., Mele, K., Vallotton, P., and Hughes, W. E. (2010) Exocytotic vesicle behaviour assessed by total internal reflection fluorescence microscopy, Traffic 11, 429-439. | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/48241 | - |
| dc.description.abstract | 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。 | zh_TW |
| dc.description.abstract | 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. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-15T06:49:55Z (GMT). No. of bitstreams: 1 ntu-100-R97450003-1.pdf: 9954059 bytes, checksum: a6d515e763ebe4b24dee3013a17b6e7f (MD5) Previous issue date: 2011 | en |
| dc.description.tableofcontents | 中文摘要 …………………………………………………………………...……...…...I
ABSTRACT ………………………………………………………………..…..……...II CONTENTS …………………………………………………………….………,,…….1 INTRODUCTION …………………………………………………………….….…....7 -- Biogenesis of secretory proteins …………………………….......................................7 -- Characteristics of the regulated secretion pathway …………………………….…......8 -- cAMP-PKA pathway is a major modulator of various secretory systems ………..….8 -- The importance and composition of salivary glands …………….………...…....……9 -- The AMP-activated protein kinase family proteins ……………………………....…10 -- The expression and functions of SIKs ……………………………………………....12 -- Hypothesis …………………………………………………………………….……..13 -- The biogenesis and composition of insulin vesicles ………….………………….….14 -- The major signaling pathway and release kinetic of insulin vesicle exocytosis …....15 MATERIALS AND METHODS ……………………………………………..….…..18 -- DNA constructs and antibodies ………………………………………………..…....18 -- Cell culture and transfection ………………………………………………...…...…18 -- Immunopreipitation and western blot analysis ……………………………...……...19 -- In vitro kinase assay …………………………………………………………..........19 -- Immunofluorescence staining, Confocal microscopy and TIRF microscopy ……....20 -- In vivo perfusion assay ……………………………………………………….......…21 -- Immunohistochemistry ………………………………………………………….…..22 RESULTS …………………………………………………………………….…….....23 -- SIK2 is specifically expressed in salivary glands ……….………………….….…....23 -- SIK2-S587 is the substrate of PKA …………………………………………..……...23 -- SIK2-pS587 is widely expressed in various LDCV-secreting glands ……….….…..28 -- SIK2-pS587 is expressed in pancreatic β cells ……………………………….….….29 -- Glucose stimulation promotes SIK2-S587 phosphorylation which is correlated with PKA activation ………………………………………………………….….….30 -- SIK2-pS587 is colocalized with PKAβ in insulin-containing LDCVs ………….….31 -- PKA/SIK2 crosstalk may be involved in the regulation of insulin vesicle translocation ………………,…………………………………………….…..……...34 DISCUSSION ……………………………………………………………...…………37 FUTURE WORK …………………………………………………………………….44 -- Part 1 : Functional studies of SIK2 ………………………………………………....44 -- Part 2 : Live-tracking and kinetics studies of insulin vesicle transport ………...…..45 -- Part 3 : The animal models for SIK2 research …………………………………..….46 REFERENCES ……………………………………………………………...…….....47 FIGURES LEGENDS ……………………………………………………...….……..55 -- SIK2 is highly expressed in salivary glands ………………………………....…...…55 -- The PKA target consensus around SIK2-S587 is highly conserved among vertebrates ……………………………………………………………….…..……...56 -- The schematic representation of SIK2 and SIK2-pS587 antibodies design and the characterization of SIK2-pS587 antibody ……………………………………...…...57 -- cAMP-PKA signaling modulates the phosphorylation of SIK2-S587 …………........58 -- Overexpression of PKAcs augments SIK2-S587 phosphorylation in vivo ……..…...59 -- The enhancement of SIK2-S587 phosphorylation is PKA dose-dependent ….......…60 -- Only PKAβ is present in immuno-complexes containing SIK2 ……….……..….….61 -- PKAβ can phosphorylate SIK2 at S587 in vitro ……………………………......…...62 -- SIK2-S587 phosphorylation occurs in salivary glands …………………..….……....63 -- A population of SIK2 expressed in thyroid and adrenal glands is phosphorylated at S587 ……….……………………………………………………………...……...64 -- SIK2 is specifically located on pancreatic islet cells corresponds to insulin staining pattern ……………………………………………………………………...…...…..65 -- Phosphorylation of SIK2 at S587 correlates with PKA activation in feeding rats ….66 -- The methodological design and the flow chart of in vivo perfusion assay ……........67 -- Glucose is sufficient to trigger SIK2-S587 phosphorylation in the fasting rats ........68 -- SIK2 colocalized with activate PKA at the peripheric rim of insulin secreting cells ……………………………………………………………………………...…..69 -- S587 phosphorylated subpopulation of SIK2 is enriched near the plasma membrane upon glucose stimulation …………………………………………………..…...…...70 -- SIK2-pS587 is colocalized with PKAβ in insulin containing granules which resemble LDCVs ….…………………………………………………………...……71 -- Glucose stimulation triggers SIK2-S587 phosphorylation and causes the augment of colocalization between SIK2-pS587 and insulin vesicles ………………..............72 -- Glucose stimulation triggers PKA/SIK2 crosstalk in insulin LDCVs …………...….73 -- SIK2 S587 phosphorylation occurs as early as in reserve pool insulin LDCVs …….74 -- PKA/SIK2 crosstalk may be involved in the regulation of insulin vesicle translocation ………………………………………………………………………....75 SUPPLEMENTARY DATA …………………………………………………....…….76 -- The schematic representation of Flag-SIK2-WT, Flag-SIK2-S587 and Flag-SIK2-KD vectors ……………………………………………………….…......76 -- The schematic representation of TIRF microscopic observation while PKA/SIK2 crosstalk regulates insulin vesicles translocation ……………………………......….77 -- The schematic representation of TIRF microscopic observation while PKA/SIK2 crosstalk regulates insulin vesicles docking .…………………………………...…...78 -- The possible role of SIK2 in regulating systemic homeostasis ………………….….79 -- The possible effect of SIK2 kinase activity and its associated proteins on the S587 residue phosphorylation ……………………………………,,,....…..…...……80 -- The phosphorylation of SIK2-S587 at the reaction of Flag-SIK2-WT with ATP only was diminished by adding selective PKA inhibitor H89………….….…....…...81 -- PKAβ is also associated with SIK2-kinase dead protein ………………….………..81 APPENDIX ………………………………………………………………….…….….82 -- The PKA dependent and PKA independent pathway of cAMP regulated vesicle exocytosis …………………………………………………………………….……..82 -- The role of SIK1 in switching on and off steroidogenic gene expression at the initial phase of ACTH stimulation ………………………………………….….…...83 -- The role of SIK2 in modulating insulin signaling in the adipocytes ……….….…....84 -- The role of SIK2 in regulating the localization of TORC2, thereby affecting CREB-dependent gene expression …………………………………….…….……..85 -- Vesicle pools in β cells …………………………………….…………….….………86 -- The signaling pathway of Glucose-stimulated insulin secretion ……….……….….87 -- Release kinetics of glucose-induced insulin secretion …………….……….….……88 -- Schematic representation of PKA structure and activation ………………..……….89 -- The expression of SIK2 in various glands which were published from Human Protein Atlas website …………………………………………………………..…...90 -- Scheme illustrating the translocation of granules from reserve pools to exocytosis ………………………………………………………………….……..…91 | |
| dc.language.iso | en | |
| dc.subject | 胰島素小泡 | zh_TW |
| dc.subject | 分泌 | zh_TW |
| dc.subject | SIK2 | zh_TW |
| dc.subject | PKA | zh_TW |
| dc.subject | β細胞 | zh_TW |
| dc.subject | secretion | en |
| dc.subject | insulin vesicles | en |
| dc.subject | β cells | en |
| dc.subject | PKA | en |
| dc.subject | SIK2 | en |
| dc.title | 探討SIK2蛋白於調控LDCVs分泌之功能角色 | zh_TW |
| dc.title | The functional role of SIK2 in regulating large dense core vesicles (LDCVs) secretion | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 99-1 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 司徒惠康,沈湯龍 | |
| dc.subject.keyword | 分泌,SIK2,PKA,β細胞,胰島素小泡, | zh_TW |
| dc.subject.keyword | secretion,SIK2,PKA,β cells,insulin vesicles, | en |
| dc.relation.page | 91 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2011-02-18 | |
| dc.contributor.author-college | 牙醫專業學院 | zh_TW |
| dc.contributor.author-dept | 口腔生物科學研究所 | zh_TW |
| 顯示於系所單位： | 口腔生物科學研究所 | |
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