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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 周涵怡 | |
| dc.contributor.author | I-I Chuo | en |
| dc.contributor.author | 卓依依 | zh_TW |
| dc.date.accessioned | 2021-06-16T17:27:11Z | - |
| dc.date.available | 2012-09-19 | |
| dc.date.copyright | 2012-09-19 | |
| dc.date.issued | 2012 | |
| dc.date.submitted | 2012-08-16 | |
| dc.identifier.citation | 1. Lodish H, Berk A, Zipursky SL, et al. (2000) Overview of the secretory pathway. Molecular Cell Biology. 4th Edition. New York: W. H. Freeman; Section 17.3.
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(2002) Membrane association domains in Ca2+-dependent activator protein for secretion mediate plasma membrane and dense-core vesicle binding required for Ca2+-dependent exocytosis, J Biol Chem. 277, 22025-34. 14. Furuichi, T., Shiraishi-Yamaguchi, Y., Sato, A., Sadakata, T., Huang, J., Shinoda, Y., Hayashi, K., Mishima, Y., Tomomura, M., Nishibe, H. & Yoshikawa, F. (2011) Systematizing and cloning of genes involved in the cerebellar cortex circuit development, Neurochemical research. 36, 1241-52. 15. Sadakata, T., Mizoguchi, A., Sato, Y., Katoh-Semba, R., Fukuda, M., Mikoshiba, K. & Furuichi, T. (2004) The secretory granule-associated protein CAPS2 regulates neurotrophin release and cell survival, The Journal of neuroscience : the official journal of the Society for Neuroscience. 24, 43-52. 16. Speidel, D., Salehi, A., Obermueller, S., Lundquist, I., Brose, N., Renstrom, E. & Rorsman, P. (2008) CAPS1 and CAPS2 regulate stability and recruitment of insulin granules in mouse pancreatic beta cells, Cell Metab. 7, 57-67. 17. Sadakata, T., Shinoda, Y., Sekine, Y., Saruta, C., Itakura, M., Takahashi, M. & Furuichi, T. (2010) Interaction of calcium-dependent activator protein for secretion 1 (CAPS1) with the class II ADP-ribosylation factor small GTPases is required for dense-core vesicle trafficking in the trans-Golgi network, J Biol Chem. 285, 38710-9. 18. Sadakata, T., Sekine, Y., Oka, M., Itakura, M., Takahashi, M. & Furuichi, T. (2012) Calcium-dependent activator protein for secretion 2 interacts with the class II ARF small GTPases and regulates dense-core vesicle trafficking, The FEBS journal. 279, 384-94. 19. Craxton, M. (2004) Synaptotagmin gene content of the sequenced genomes, BMC genomics. 5, 43. 20. Gauthier, B. R. & Wollheim, C. B. (2008) Synaptotagmins bind calcium to release insulin, Am J Physiol Endocrinol Metab. 295, E1279-86. 21. 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(2007) Synaptotagmin-12, a synaptic vesicle phosphoprotein that modulates spontaneous neurotransmitter release, J Cell Biol. 176, 113-24. 26. Hosaka, M., Suda, M., Sakai, Y., Izumi, T., Watanabe, T. & Takeuchi, T. (2004) Secretogranin III binds to cholesterol in the secretory granule membrane as an adapter for chromogranin A, J Biol Chem. 279, 3627-34. 27. Hosaka, M., Watanabe, T., Yamauchi, Y., Sakai, Y., Suda, M., Mizutani, S., Takeuchi, T., Isobe, T. & Izumi, T. (2007) A subset of p23 localized on secretory granules in pancreatic beta-cells, The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society. 55, 235-45. 28. Rasmussen, C. & Garen, C. (1993) Activation of calmodulin-dependent enzymes can be selectively inhibited by histone H1, J Biol Chem. 268, 23788-91. 29. Huber, L. A. (2003) Organelle Proteomics: Implications for Subcellular Fractionation in Proteomics, Circulation Research. 92, 962-968. 30. Yi, Z., Yokota, H., Torii, S., Aoki, T., Hosaka, M., Zhao, S., Takata, K., Takeuchi, T. & Izumi, T. (2002) The Rab27a/Granuphilin Complex Regulates the Exocytosis of Insulin-Containing Dense-Core Granules, Molecular and Cellular Biology. 22, 1858-1867. 31. Tixier-Vidal, A., Barret, A., Picart, R., Mayau, V., Vogt, D., Wiedenmann, B. & Goud, B. (1993) The small GTP-binding protein, Rab6p, is associated with both Golgi and post-Golgi synaptophysin-containing membranes during synaptogenesis of hypothalamic neurons in culture, J Cell Sci. 105 ( Pt 4), 935-47. 32. Wasmeier, C. & Hutton, J. C. (1999) Secretagogue-dependent phosphorylation of phogrin, an insulin granule membrane protein tyrosine phosphatase homologue, Biochem J. 341 ( Pt 3), 563-9. 33. Horike, N., Takemori, H., Katoh, Y., Doi, J., Min, L., Asano, T., Sun, X. J., Yamamoto, H., Kasayama, S., Muraoka, M., Nonaka, Y. & Okamoto, M. (2003) Adipose-specific expression, phosphorylation of Ser794 in insulin receptor substrate-1, and activation in diabetic animals of salt-inducible kinase-2, J Biol Chem. 278, 18440-7. 34. Yamagata, K., Senokuchi, T., Lu, M., Takemoto, M., Fazlul Karim, M., Go, C., Sato, Y., Hatta, M., Yoshizawa, T., Araki, E., Miyazaki, J. & Song, W. J. (2011) Voltage-gated K+ channel KCNQ1 regulates insulin secretion in MIN6 beta-cell line, Biochem Biophys Res Commun. 407, 620-5. 35. Daily, N. J., Boswell, K. L., James, D. J. & Martin, T. F. (2010) Novel interactions of CAPS (Ca2+-dependent activator protein for secretion) with the three neuronal SNARE proteins required for vesicle fusion, J Biol Chem. 285, 35320-9. 36. Lee, B. H., Min, X., Heise, C. J., Xu, B. E., Chen, S., Shu, H., Luby-Phelps, K., Goldsmith, E. J. & Cobb, M. H. (2004) WNK1 phosphorylates synaptotagmin 2 and modulates its membrane binding, Molecular cell. 15, 741-51. 37. Handa, N., Takagi, T., Saijo, S., Kishishita, S., Takaya, D., Toyama, M., Terada, T., Shirouzu, M., Suzuki, A., Lee, S., Yamauchi, T., Okada-Iwabu, M., Iwabu, M., Kadowaki, T., Minokoshi, Y. & Yokoyama, S. (2011) Structural basis for compound C inhibition of the human AMP-activated protein kinase alpha2 subunit kinase domain, Acta crystallographica Section D, Biological crystallography. 67, 480-7. 38. Hwang, C. S., Shemorry, A. & Varshavsky, A. (2010) N-terminal acetylation of cellular proteins creates specific degradation signals, Science. 327, 973-7. 39. Sadakata, T., Washida, M. & Furuichi, T. (2007) Alternative splicing variations in mouse CAPS2: differential expression and functional properties of splicing variants, BMC neuroscience. 8, 25. 40. Nojiri, M., Loyet, K. M., Klenchin, V. A., Kabachinski, G. & Martin, T. F. (2009) CAPS activity in priming vesicle exocytosis requires CK2 phosphorylation, J Biol Chem. 284, 18707-14. 41. Narendran, P., Estella, E. & Fourlanos, S. (2005) Immunology of type 1 diabetes, QJM : monthly journal of the Association of Physicians. 98, 547-56. 42. Prentki, M. & Nolan, C. J. (2006) Islet beta cell failure in type 2 diabetes, J Clin Invest. 116, 1802-12. 43. Kumagai, A., Horike, N., Satoh, Y., Uebi, T., Sasaki, T., Itoh, Y., Hirata, Y., Uchio-Yamada, K., Kitagawa, K., Uesato, S., Kawahara, H., Takemori, H. & Nagaoka, Y. (2011) A potent inhibitor of SIK2, 3, 3', 7-trihydroxy-4'-methoxyflavon (4'-O-methylfisetin), promotes melanogenesis in B16F10 melanoma cells, PLoS One. 6, e26148. | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/64030 | - |
| dc.description.abstract | 內分泌及外分泌腺體參與體內各種不同器官的相互調控進而影響身體的發育、生殖、代謝及動態恆定等等，對於生命體來說扮演非常重要的角色。 分泌系統的異常或缺陷會導致代謝紊亂，例如乾口症及糖尿病等代謝症候群疾病，嚴重者則會危及生命。
許多消化酵素、神經生肽或荷爾蒙等分泌型蛋白會被包裹在大型緻密核心囊泡 (large dense core vesicle, LDCV)中並進行運輸及釋放。 新生成的LDCV會被運送到細胞質中暫時儲存，稱為儲存型囊泡庫 (reserve pool, RP)或黏附在細胞膜上等待釋放的立即釋放型囊泡庫 (readily releasable pool, RRP)，兩者的動態平衡是經由細胞內部和外部的訊息來嚴格控制，然而決定儲存到RP或運送到RRP的詳細分子機制，目前仍尚在研究當中。 SIK2屬於AMPK激酶家族中的一員。根據先前我們實驗室的研究顯示，SIK做為一個PKA調控LDCV分泌的下游因子。 不同激酶活性的SIK2主要存在於不同的LDCV囊泡庫中，而利用SIK2激酶活性抑制劑處理後發現會促使LDCV的釋放增加，暗示著SIK2可能藉由其激酶活性來調控LDCV的分泌。 本篇我們利用生化分餾(biochemical fractionation)的方式分離Rinm5F胰臟癌細胞的胰島素囊泡 (insulin-containing LDCVs)，確認SIK2確實位在胰島素囊泡上。 接著以SIK2已知的磷酸化共同基因序列 (phosphorylation consensus motif)進行電腦資料運算比對，鎖定CAPS2 (Calcium-dependent secretion activator 2)和Syt12 (Synaptotagmin XII)可能是SIK2調控的下游因子。 我們證明了CAPS2和Syt12與SIK2共同存在於相同的fraction，且CAPS2與SIK2確實存在於同一個複合體，但詳細的功能仍需進一步的研究。 綜合以上結果，我們藉由SIK2激酶活性與LDCV動態的關係，並確定其可能的下游分子，將有助於建立SIK2在調控LDCV分泌過程中所扮演的角色。 | zh_TW |
| dc.description.abstract | Endocrine and exocrine secretory glands are important controls of the organism’s homeostasis, by communicating and coordinating among the different organ systems of the body. Defects in these secretory processes can result in life threatening diseases, while minor deregulations can cause metabolic disorders such as diabetes mellitus and xerostomia. Secretion through the large dense-core vesicles (LDCVs) is the primary route for the release of neuropeptides. In the cell, newly synthesized LDCVs are transported and temporarily stored as reserve pool (RP) or readily releasable pool (RRP) vesicles, and this dynamic equilibrium is tightly controlled by both internal and external clues. However, the molecular mechanism underlying the decision for transport and storage as RP or RRP vesicles is currently unknown. SIK2 (salt-inducible kinase 2) is a family member of the AMPK serine/threonine kinases. Previous data from our lab indicate that SIK2 is a LDCV resident and serves as substrate for PKA kinase activity, which is a major trigger of LDCVs secretion. Subsets of SIK2 proteins presenting differential kinase activities localized to distinct pools of LDCVs, while treatment with specific inhibitor of SIK2 kinase activity promotes LDCV release, suggesting that SIK2 may regulate LDCV secretion through its kinase activity. Here, we perform biochemical fractionation of LDCVs from Rinm5F insulinoma cells by sucrose gradient centrifugation, and confirm the cofractionation of SIK2 with insulin vesicles. Then, we use in silico data mining to identify Calcium-dependent secretion activator 2 (CAPS2) and Synaptotagmin XII (Syt12) as candidate substrates of SIK2 kinase activity by conservation of phosphorylation motif consensus. CAPS2 and Syt12 cofractionate with SIK2, while CAPS2 present in the SIK2-containing complex. Whether CAPS2 and Syt12 display functional association with SIK2 is under study. In parallel, we assay how different post-translational modifications affect on SIK2 kinase activity in vitro, using recombinant Syntide sequence as substrate. Together, these studies will help establish the role of SIK2 in regulating LDCV secretion, by relating the status of its kinase activity with LDCV dynamics, and identifying its putative downstream effector molecules. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-16T17:27:11Z (GMT). No. of bitstreams: 1 ntu-101-R99450001-1.pdf: 14513991 bytes, checksum: d503e9338b610b2a6db20bc8e5b84cc1 (MD5) Previous issue date: 2012 | en |
| dc.description.tableofcontents | 口試委員會審定書 ........................................... I
致謝 .................................................... II 中文摘要 ................................................. IV ABSTRACT ............................................... VI CONTENTS ................................................ 1 INTRODUCTION ............................................ 6 - The role of secretory system .......................... 6 - Characteristic of regulated secretory pathway in pancreatic β cells .................................... 7 - The expression and function of SIK2 in insulin-containing LDCV secretion ........................................ 8 - The distinctive pools of insulin-containing LDCV in secretion ............................................ 10 - Hypothesis ........................................... 11 - CAPS family proteins involved in trafficking and exocytosis of LDCV secretion ......................... 11 - Syt family involved in Ca2+-dependent or –independent triggering LDCV secretion ............................ 13 MATERIALS AND METHODS .................................. 15 - Cell culture and transfection ........................ 15 - DNA constructs and antibodies ........................ 15 - Immunofluorescence staining .......................... 16 - Biochemical fractionation of insulin-containing LDCV.. 17 - ELISA measurement of insulin-containing LDCV ......... 18 - In silico data mining of SIK2 putative substrates .... 18 - Immunoprecipitation and Western blot analysis ........ 19 - In vitro kinase assay ................................ 20 - Bacterial expression and purification of GST-Syntide-2 fusion protein ....................................... 21 - RNA extraction and RT-PCR ............................ 22 RESULTS ................................................ 23 - SIK2 cofractionates with insulin-containing LDCV ..... 23 - SIK2 with differential kinase activities cofractionate with distinct pools of insulin vesicles .............. 24 - Identification of putative substrates of SIK2 by in silico data mining .......................................... 25 - Both CAPS2 and Syt12 cofractionate with SIK2 ......... 28 - CAPS2 is present in the same immunocomplex containing SIK2 ...................................................... 29 - SIK2 phosphorylates CAPS2 at threonine residues ...... 29 - Autophosphorylation and kinase activity of SIK2 ...... 30 DISCUSSION ............................................. 32 - The post-translational modification of SIK2 .......... 32 - CAPS2c isoform may specifically associate with SIK2 .. 34 - The possible effects of SIK2 phosphorylation on CAPS2 and Syt12 in insulin secretion ........................... 34 - Future perspectives SIK2 regulation in clinical applications ......................................... 36 REFERENCES ............................................. 38 FIGURE LEGENDS ......................................... 47 - Biochemical fractionation of insulin-containing LDCV in Rinm5F cells ........................................... 47 - Different kinase activities of SIK2 cofractionate with distinct pools of insulin- containing LDCVs .......... 48 - In silico search for putative SIK2 phosphorylation targets ...................................................... 49 - Putative SIK2 substrate proteins containing multiple optimally match sites ................................ 50 - CAPS2 and Syt12 as putative SIK2 substrates .......... 51 - Sequence context around the putative SIK2 target sites in CAPS2 and Syt12 are highly conserved among vertebrate species .............................................. 52 - CAPS2 and Syt12 cofractionate with SIK2 in insulin- containing LDCV ...................................... 53 - CAPS2 is present in the immunocomplex of SIK2 ........ 54 - In vitro phosphorylation of SIK2 on CAPS2 at Threonine residues ............................................. 55 - Autophosphorylation of SIK2 at the T175 residue ...... 56 APPENDIX ............................................... 57 - The protein sequence and secondary structure around the target consensus of PKA in SIK2 are highly conserved in vertebrates .......................................... 57 - SIK2 and SIK2 pS587 are widely expressed in LDCV-secreting glands ............................................... 58 - Inhibition of SIK2 kinase activity by treatment with Compound C induces insulin release in Rinm5F cells ... 59 - SIK2 is colocalized with insulin-containing LDCVs .... 60 - Subsets of SIK2 proteins presenting differential kinase activities localized to distinct pools of insulin- containing LDCVs ..................................... 61 - Overexpression of SIK2 induced CAPS2 mRNA expression.. 62 - SIK2 regulates the localization of TORC2, thereby mediating CREB activation and affecting CREB-dependent gene expression ...................................... 63 - SIK2 is degraded after Compound C treatment .......... 64 - SDS-PAGE gel analysis of GST-Syntide-2 purification .. 65 - Acetylation sites of SIK2 at kinase domain ........... 66 - Alternative splicing variations in mouse CAPS2 ....... 67 - Design of GST recombinant protein construct .......... 68 - CAPS2 siRNA form National RNAi Core Facility Platform ...................................................... 69 - SIK2 sites on Syt12 reside on the solute interface ... 70 | |
| dc.language.iso | en | |
| dc.subject | 分泌 | zh_TW |
| dc.subject | SIK2 | zh_TW |
| dc.subject | LDCV | zh_TW |
| dc.subject | 激酶 | zh_TW |
| dc.subject | 活性 | zh_TW |
| dc.subject | 下游因子 | zh_TW |
| dc.subject | CAPS2 | zh_TW |
| dc.subject | LDCVs | en |
| dc.subject | secretion | en |
| dc.subject | SIK2 | en |
| dc.subject | CAPS2 | en |
| dc.subject | kinase activity | en |
| dc.subject | substrate | en |
| dc.title | 鑑定SIK2蛋白激酶調控LDCV分泌的下游因子 | zh_TW |
| dc.title | Identification of SIK2 downstream substrates for regulating large dense-core vesicles (LDCVs) secretion | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 100-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 沈湯龍,司徒惠康 | |
| dc.subject.keyword | 分泌,SIK2,LDCV,激酶,活性,下游因子,CAPS2, | zh_TW |
| dc.subject.keyword | secretion,SIK2,LDCVs,kinase activity,substrate,CAPS2, | en |
| dc.relation.page | 70 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2012-08-16 | |
| 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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