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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 莊寧寧(Nin-Nin Chuang) | |
| dc.contributor.author | LING-YUN CHU | en |
| dc.contributor.author | 儲凌雲 | zh_TW |
| dc.date.accessioned | 2021-06-13T16:26:39Z | - |
| dc.date.available | 2005-07-26 | |
| dc.date.copyright | 2005-07-26 | |
| dc.date.issued | 2005 | |
| dc.date.submitted | 2005-07-15 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/38119 | - |
| dc.description.abstract | 在本實驗中，我們從處於分裂期的斑馬魚胚胎分離出作用中的KSR (Kinase Suppressor of Ras)蛋白複合體，在純化KSR的過程中，我們發現RACK1 (Receptor for Activated C Kinase 1) 和KSR之間可能有交互作用存在，經免疫沉澱法證實RACK1存在於斑馬魚KSR蛋白複合體中。為了探討KSR與RACK1的關係，我們使用大腸桿菌表現的重組RACK1進行實驗，重組14-3-3β作為平行實驗，分別以大腸桿菌表現的重組KSR與從斑馬魚胚胎純化的KSR，透過RACK1或14-3-3β的親和力管柱(affinity column)進行結合測試，我們發現大腸桿菌表現出的重組KSR蛋白無法與RACK1或14-3-3β親和力管柱結合，而從斑馬魚胚胎純化的KSR則可以。將純化的斑馬魚KSR與重組RACK1先進行結合反應後，我們發現RACK1與KSR的結合會干擾KSR與14-3-3親和力管柱的結合，因此RACK1在KSR上的作用可能和KSR的位置轉移有關。為了探討RACK1在細胞中是否與KSR的位置轉移有關，我們使用小鼠細胞株BALB/3T3進行實驗，利用免疫沉澱法我們發現RACK1只在10%胎牛血清培養的BALB/3T3細胞中與KSR結合，而在缺乏10%胎牛血清培養24小時的細胞中則不會結合。在進行免疫沉澱的實驗中，我們發現斑馬魚KSR與RACK1的結合需要磷酸的參與，而在BALB/3T3細胞中，此交互作用部份需要磷酸參與，部分則不需要。我們使用RACK1的siRNA抑制RACK1在細胞中的表現，72小時後RACK1的表現量約為正常量的一半。我們發現由表皮生長激素 (EGF) 所引起的KSR位置轉移會因為RACK1表現的抑制而受到干擾。綜合這些結果，我們認為RACK1可能具有協助KSR在細胞中進行位置轉移的作用，並且因此調控細胞的生長與分裂。 | zh_TW |
| dc.description.abstract | In this study, functional KSR (Kinase Suppressor of Ras) complex was isolated from zebrafish embryos at cleavage stage. During the process of KSR purification, we found that there may be an interaction between RACK1 (Receptor for Activated C Kinase 1) and KSR. It was demonstrated that RACK1 is a member of functional KSR complex by immunoprecipitation of KSR. To study the interaction between KSR and RACK1, E. coli expressed recombinant RACK1 was used and the recombinant 14-3-3β was used for parallel study. Both E. coli expressed KSR and the purified zebrafish KSR were subjected to affinity chromatography using RACK1 or 14-3-3β affinity column. We found that E. coli expressed recombinant KSR did not interact with RACK1 or 14-3-3β affinity column, while the purified zebrafish KSR did. The purified zebrafish KSR was incubated with RACK1 and we found that the binding of RACK1 on KSR disturbed the interaction of KSR with 14-3-3β affinity column. It suggests that RACK1 may be a protein related to the translocation of KSR. To elucidate the role of RACK1 in translocation of KSR, a mouse cell line, BALB/3T3, was used to study. Using the immunoprecipitation of KSR, we found that RACK1 interacted with KSR only in cells cultured with 10% FBS but not in cells starved in serum-free medium for 24 hours. During the immunoprecipitation study, we found that the interaction between RACK1 and KSR is phosphate dependent in zebrafish and this interaction is partly phosphate-dependent and partly phosphate-independent in BALB/3T3 cells. Mouse RACK1 siRNA was used to downregulate the expression of RACK1 in BALB/3T3 cells and the expression of RACK1 was about half of the normal amount after 72 hours. We found that the EGF-induced translocation of KSR was suppressed in RACK1 downregulated cells. Altogether the data, RACK1 may be a protein to facilitate the translocation of KSR and it suggests that RACK1 has a role in regulating cell growth and proliferation. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-13T16:26:39Z (GMT). No. of bitstreams: 1 ntu-94-R92b41015-1.pdf: 1600727 bytes, checksum: ade5bd1d864e89d972a19388f938af84 (MD5) Previous issue date: 2005 | en |
| dc.description.tableofcontents | Contents
Abstract…………………………………………………………………………... 1 Introduction……………………………………………………………………... 3 ERK/ MAPK pathway………………………………………………………….. 3 Scaffolding proteins…………………………………………………………….. 3 Kinase Suppressor of Ras (KSR)……………………………………………….. 4 The translocation of KSR………………………………………………………. 6 14-3-3 proteins………………………………………………………………….. 6 Receptor for Activated C Kinase 1 (RACK1)………………………………….. 7 Materials and Reagents………………………………………………………. 10 Bio-materials……………………………………………………………………. 10 Materials………………………………………………………………………… 10 Instruments……………………………………………………………………… 10 Reagents………………………………………………………………………… 11 Primers………………………………………………………………………….. 15 E. coli competent cells………………………………………………………….. 15 Methods…………………………………………………………………………... 17 Cell culture……………………………………………………………………… 17 RNA extraction and the first strand cDNA Synthesis…………………………... 17 Polymerase Chain Reaction (PCR) for mouse ksr amplification……………….. 17 Agarose gel electrophoresis and ethidium bromide (EtBr) staining……………. 18 Gel extraction of DNA fragments………………………………………………. 18 Directional TA cloning and Transformation……………………………………. 19 Plasmids extraction……………………………………………………………... 19 Restriction Enzyme digestion…………………………………………………... 20 DNA Ligation…………………………………………………………………... 20 Construction of KSR expression plasmid for E. coli expression……………….. 20 Construction of nLumio-KSR Fusion protein expression plasmid for mammalian cell expression…………………………………………………. 21 Transfection……………………………………………………………………... 21 Mouse RACK1 siRNA synthesis and the downregulation of RACK1…………. 22 Subcellular fractionation………………………………………………………... 22 Fluorescence microscopy……………………………………………………….. 23 Purification of KSR from Zebrafish embryos…………………………………... 23 Immunoprecipitation of KSR complex from zebrafish embryos and cells……... 25 Production of mouse RACK1-encoded, 14-3-3β-encoded and KSR-encoded fusion proteins in bacteria…………………………………………………... 26 Preparation and using of HiTrap-PEPTaxol, RACK1 and 14-3-3β affinity columns……………………………………………………………………... 27 Using of HiTrap chelating affinity column……………………………………... 28 Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE)…… 28 Western immunoblotting……………………………………………………….. 29 Silver-staining…………………………………………………………………... 29 Quantitation of protein………………………………………………………….. 30 Results…………………………………………………………………………….. 31 KSR was isolated from zebrafish embryos. ……………………………………. 31 Zebrafish KSR was further purified by HiTrap-PEPTaxol affinity column and HiTrap-14-3-3β affinity column and RACK1 is a member of zebrafish KSR complex……………………………………………….......................... 31 Mouse ksr was cloned for E. coli expression…………………………………… 33 E. coli expressed KSR did not interact with recombinant RACK1 or recombinant 14-3-3β, while purified zebrafish KSR did.…………………... 33 Recombinant RACK1 interacted with zebrafish KSR and disturbed the interaction between zebrafish KSR and recombinant 14-3-3β……………... 34 KSR only interacted with RACK1 in serum stimulated BALB/3T3 cells but not in starved cells……………………………………………………………… 35 The expression of RACK1 in BALB/3T3 cells was downregulated by RACK1 siRNA. ……………………………………………………………………… 35 The amount of KSR in the membrane fractions was induced by EGF stimulation and the EGF induction was interfered by RACK1 downregulation in BALB/3T3 cells………………………………………… 36 The Lumio reagent is not applicable in labeling the location of KSR overexpressed in BALB/3T3 cells………………………………………….. 36 Discussion................................................................................................................ 38 Summary…………………………………………………………………………. 44 References………………………………………………………………………... 45 Figures……………………………………………………………………………. 55 Supplementary depiction……………………………………………………... 85 | |
| dc.language.iso | en | |
| dc.subject | RACK1 | zh_TW |
| dc.subject | KSR | zh_TW |
| dc.title | RACK1與KSR的交互作用與其細胞生理意義之研究 | zh_TW |
| dc.title | The functional role of RACK1 in the molecular translocation of KSR in cells | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 93-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 陳俊宏(Jiun-Hong Chen),李心予(Hsinyu Lee),潘建源(Chien-Yuan Pan),黃偉邦(Wei-Pang Huang) | |
| dc.subject.keyword | RACK1,KSR, | zh_TW |
| dc.relation.page | 86 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2005-07-15 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 動物學研究研究所 | zh_TW |
| 顯示於系所單位： | 動物學研究所 | |
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