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dc.contributor.advisor이종란-
dc.contributor.author임효진-
dc.creator임효진-
dc.date.accessioned2016-08-26T11:08:08Z-
dc.date.available2016-08-26T11:08:08Z-
dc.date.issued2009-
dc.identifier.otherOAK-000000051653-
dc.identifier.urihttps://dspace.ewha.ac.kr/handle/2015.oak/201845-
dc.identifier.urihttp://dcollection.ewha.ac.kr/jsp/common/DcLoOrgPer.jsp?sItemId=000000051653-
dc.description.abstractAdaptor proteins play a central role in lymphocyte activation by mediating intermolecular interactions and assembling signaling complexes. Present studies show new function of two adaptor proteins, Odin and BLNK in T and B lymphocytes, respectively. Odin was identified as one of the tyrosine phosphorylated proteins recruited to lipid raft after TCR ligation in Jurkat T cell lines. Using the Jurkat T cells in which Odin expression was reduced by the introduction of siRNA, it has been shown that Odin did not play a major role in the initial signaling pathways after TCR activation, but enhanced TCR-induced activation of transcription factors, NF-AT and NF-κB. Actin polymerization in the contact sites between T cells and antigen presenting cells was increased in the Jurkat T cells with reduced Odin expression. Moreover, adhesion to fibronectin and Rap1 activation was enhanced in these Jurkat T cells with reduced Odin expression. These results suggest that Odin may negative regulation basal integrin activation, as well as TCR stimulation-dependent ‘inside-out’ signaling. Another adaptor protein, BLNK is a homolog of SLP-76 and is expressed in B cells. In this thesis, the binding motif of BLNK required for interaction with phosphatidylinositide3-kinase p85 subunit was defined, using the GST-p85 N-SH2 fusion protein and various deletion and point mutants of BLNK. From these experiments, phosphorylation of 72 and/or 84 tyrosine residue(s) at the N-terminus of BLNK was shown to be required for the interaction with the p85 N-SH2 domain. This association of BLNK with the p85 was also detected in a B cell line, WEHI 231 after the ligation of BCR. These data suggest that BLNK may play a role in BCR stimulation-dependent PI3K activation through association with the p85 subunit in a tyrosine phosphorylation-dependent manner.;Adaptor 단백질들은 다른 단백질과의 상호 결합을 통해서 임파구 세포의 세포막에서 전달되는 활성화 신호를 세포 내로 전달하는 역할을 수행한다. 본 연구에서는 Odin과 BLNK 라는 두 종류의 adaptor 단백질의 기능을 T 임파구와 B 임파구에서 연구하였다. Odin은 T 임파구 수용체 자극에 의해 lipid rafts로 이동하여 인산화 된 후 또 다른 adaptor 단백질들인 LAT과 SLP-76에 결합함을 확인하였다. 또한 si RNA 발현을 통하여 Odin의 발현이 감소된 Jurkat T 임파구 이용 실험결과, Odin 단백질은 T 임파구 수용체 자극에 의한 초기 신호전달에는 크게 영향을 미치지 않으나 integrin 활성화에 의한 adhesion 및 NF-AT과 NF-κB와 같은 전사조절인자들의 활성화에 영향을 미치는 것이 확인되었다. 즉, Odin 단백질의 발현이 억제되었을 때 항원수용체 자극에 의한 전사조절인자의 활성화가 증가되었고, Rap1의 활성화, integrin에 의한 adhesion및 F-actin clustering 등이 항원수용체 자극에 관계없이 증가됨을 관찰하였다. 이러한 결과들은 Odin 단백질이 항원수용체 자극에 의한 ‘inside-out’ 신호전달 과정에서 중요한 역할을 수행할 가능성을 제시한다. 또 다른 adaptor 단백질, BLNK는 SLP-76와 유사한 domain 구조를 가지며 B 임파구 항원수용체 자극에 의해 tyrosine 인산화가 일어남이 보고된 바 있다. 본 연구에서는 BLNK와 p85의 결합에 관계하는 motif를 mapping하였다. 즉 분석하였다. 다양한 BLNK 변형 유전자를 발현시킨 세포주에서의 실험결과, BLNK는 72번과 84번의 tyrosine의 인산화를 통하여 p85의 N-SH2 영역과 결합함을 확인했다. 이러한 BLNK-p85의 상호결합은 WEHI 231 B 임파구에서도 항원수용체 자극 후에 관찰되었다. 이러한 결과들은 BLNK 단백질이 또한 B 임파구 수용체 자극에 의한 PI3K 신호전달을 매개할 가능성을 제시한다.-
dc.description.tableofcontentsⅠ. Introduction = 1 Ⅱ. Materials and Methods = 11 1. Abs and reagents = 11 2. Cell culture and T cell, B cell activation = 12 3. Plasmids and cloning = 12 4. Transient expression of plasmid DNA = 14 5. Measurements of intracellular Ca^(2+) = 14 6. Immunoprecipitation and immunoblotting = 15 7. Luciferase assay = 15 8. T cell-APC conjugation assay = 16 9. Adhesion assays = 16 10. Rap1 activation assays = 17 11. Precipitation of GST-fusion proteins = 17 Ⅲ. Results = 18 1. A functional study of an adaptor protein, Odin in T lymphocytes = 18 1.1 Identification of Odin, a signaling molecule recruited to lipid raft after TCR-ligation = 18 1.2 TCR-mediated signaling study in Odin gene knock-down Jurkat T cells = 21 1.3 Effects of Odin in cell adhesion by integrin binding = 24 2. Tyrosine-phosphorylation-dependent interaction of BLNK with the p85 subunit of phosphoinositide 3-Kinase = 34 2.1 Tyrosine-phosphorylated BLNK associates with the amino-terminal SH2 domain of p85 = 34 2.2 Tyrosine phosphorylation of BLNK at position 72 and/or 84 mediates the interaction with the N-SH2 domain of p85 = 38 2.3 Tyrosine-phosphorylated BLNK associates in vivo with the N-SH2 of p85 = 40 Ⅳ. Discussion = 45 Ⅴ. References = 49 Ⅵ. 국문초록 = 55-
dc.formatapplication/pdf-
dc.format.extent1359798 bytes-
dc.languageeng-
dc.publisher이화여자대학교 대학원-
dc.titleFunctional Studies of Two Adaptor Proteins, Odin and BLNK in T and B Lymphocytes-
dc.typeMaster's Thesis-
dc.format.pageⅵ, 56 p.-
dc.identifier.thesisdegreeMaster-
dc.identifier.major대학원 생명·약학부생명과학전공-
dc.date.awarded2009. 2-
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