Identification of SIK2 downstream substrates for regulating large dense-core vesicles (LDCVs) secretion
碩士 === 國立臺灣大學 === 口腔生物科學研究所 === 100 === 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 m...
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ndltd-TW-100NTU055920052015-10-13T21:50:17Z http://ndltd.ncl.edu.tw/handle/00111764696896895957 Identification of SIK2 downstream substrates for regulating large dense-core vesicles (LDCVs) secretion 鑑定SIK2蛋白激酶調控LDCV分泌的下游因子 I-I Chuo 卓依依 碩士 國立臺灣大學 口腔生物科學研究所 100 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. 周涵怡 2012 學位論文 ; thesis 70 en_US |
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碩士 === 國立臺灣大學 === 口腔生物科學研究所 === 100 === 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.
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author2 |
周涵怡 |
author_facet |
周涵怡 I-I Chuo 卓依依 |
author |
I-I Chuo 卓依依 |
spellingShingle |
I-I Chuo 卓依依 Identification of SIK2 downstream substrates for regulating large dense-core vesicles (LDCVs) secretion |
author_sort |
I-I Chuo |
title |
Identification of SIK2 downstream substrates for regulating large dense-core vesicles (LDCVs) secretion |
title_short |
Identification of SIK2 downstream substrates for regulating large dense-core vesicles (LDCVs) secretion |
title_full |
Identification of SIK2 downstream substrates for regulating large dense-core vesicles (LDCVs) secretion |
title_fullStr |
Identification of SIK2 downstream substrates for regulating large dense-core vesicles (LDCVs) secretion |
title_full_unstemmed |
Identification of SIK2 downstream substrates for regulating large dense-core vesicles (LDCVs) secretion |
title_sort |
identification of sik2 downstream substrates for regulating large dense-core vesicles (ldcvs) secretion |
publishDate |
2012 |
url |
http://ndltd.ncl.edu.tw/handle/00111764696896895957 |
work_keys_str_mv |
AT iichuo identificationofsik2downstreamsubstratesforregulatinglargedensecorevesiclesldcvssecretion AT zhuōyīyī identificationofsik2downstreamsubstratesforregulatinglargedensecorevesiclesldcvssecretion AT iichuo jiàndìngsik2dànbáijīméidiàokòngldcvfēnmìdexiàyóuyīnzi AT zhuōyīyī jiàndìngsik2dànbáijīméidiàokòngldcvfēnmìdexiàyóuyīnzi |
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1718069131589189632 |