臺灣博碩士論文加值系統

E-Ras主要表現在未分化的胚胎幹細胞中，跟一般的Ras蛋白質序列相比，E-Ras蛋白質本身即具備致癌Ras的蛋白質突變胺基酸，可維持GTP-locked，並在N端有一段多餘的序列。E-Ras主要是以GTP結合的形式使細胞轉型。本研究建立了E-ras穩定轉染的BALB/3T3纖維母細胞株，並探討caveolin-1在E-ras轉染細胞中所扮演的功能。利用Triton X-114分萃膜蛋白後，發現caveolin-1只分布在疏水層，而在轉型細胞中其表現則較少；caveolin-2表現量則持平，並有些分佈到親水層；此外，有很多RasGAP集中在Triton X-114親水層。利用蔗糖梯度超高速離心法處理後，發現caveolae的密度在E-ras轉染的細胞中會增加。這種現象可以直接利用大腸桿菌表現的重組E-Ras及KB-Ras蛋白質於in vitro實驗中再現。因此，我們提出E-Ras和KB-Ras可直接經由細胞質傳送到caveolae中，並造成caveolae密度增加。

E-Ras is expressed in undifferentiated embryonic stem (ES) cells. E-Ras proteins have mutation sites responsible for trapping GTP as oncogenic Ras, plus an extra N-terminal sequences which is not found in conserved Ras protein sequences. E-Ras proteins are mainly GTP-bounded and easily lead to cellular transformation. We have established stable clones of BALB/3T3 cells transfected with E-ras and used this cell line to study the functional role of the caveolin-1 upon cells transfected with E-ras. Using Triton X-114 to fractionate membrane proteins, caveolin-1 was exclusively clustered in Triton X-114-B (hydrophobic phase), with decreased amounts in cells transformed with E-ras, whereas caveolin-2 exhibited no difference of amounts in both Triton X-114-H (hydrophilic phase) and Triton X-114-B. Furthermore, RasGAP, the GTPase activator protein occurred mainly in Triton X-114-H. The expression of RasGAP was significant in Triton X-114-H upon transformation with E-ras. Of interest, caveolae was founded with heavier density upon transformation with E-ras as revealed wtih sucrose gradient ultracentrifugation. This transition of density in caveolae could be reconstituted with recombinant E-Ras proteins. The finding was confirmed with recombinant KB-Ras(Q61K) proteins in cells without transformation. Therefore, E-Ras and KB-Ras(Q61K) may share a similar route from cytosol to caveolae.

口試委員會審定書 I
誌謝 II
中文摘要 III
Abstract IV
Table of contents VI
Index of figures VIII
Introduction 1
General properties of Ras protein 1
Tertiary structure and conserved nucleotide binding motif of Ras protein 1
C-terminus of Ras protein for membrane localization and transformation 3
Characteristics of E-Ras 5
Regulatory role of RasGAP 6
General properties of caveolins 7
Caveolins and cell transformation 8
Materials and Methods 10
Reagents 10
Cell culture 11
Generation of E-ras constructs 12
Transfection of E-ras 12
Identification of E-Ras protein expression by in vivo staining of tetracysteine tag 13
Anchorage-independent growth assay 14
Sucrose gradient ultracentrifugation 14
Immunfluorescence microscopy of caveolin-1 15
Membrane isolation 16
Phase separation of membrane pellet proteins with Triton X-114 16
Immunoprecipitaion of caveolin-1, PY27-caveolin-2 or RasGAP from Triton-X 114 phase fractions 17
Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) 18
Western immunoblotting analysis 18
Results 20
Expression of E-Ras proteins in BALB/3T3 cells transfected with E-ras 20
Enhanced anchorage-independent growth in cells transfected with E-ras 20
Downregulation of caveolin-1 and overexpression of RasGAP upon cells transfected with E-ras 21
Redistribution of caveolae to higher sucrose density in cells transfected with E-ras 22
Location of caveolin-2 in sucrose gradient in cells transfected with E-ras 22
Location and pattern of caveolin-1 in cells transfected with E-ras 23
Incorporation of Ras proteins into caveolae 23
Recruitment of caveolin-2 with RasGAP 24
Discussion 25
Transformation of cells transfected with E-ras 25
Different expression pattern of caveolin-1 and caveolin-2 upon cells transfected with E-ras 25
Redistribution of caveolae to heavier density upon cells transfected with E-ras 26
Incorporation of E-Ras to caveolae 27
Interaction with RasGAP and caveolin-2 28
References 30

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