臺灣博碩士論文加值系統

在本實驗中，我們利用重組蛋白和親和管柱進行細胞外(in vitro)的實驗，發現重組syndecan-2和RACK1有交互作用，而在細胞內(in vivo)實驗，經免疫沉澱法和細胞免疫染色法也證實RACK1的確存在於syndecan-2蛋白複合體中，且兩分子的交互作用主要位於細胞膜上。進一步探討syndecan-2和RACK1的作用，我們發現當syndecan-2第180位置的酪胺酸在磷酸化狀態下，和RACK1的交互作用有增強的現象。
另一方面，我們以已知會造成syndecan-2酪氨酸磷酸化的細胞素(cytokine)，巨噬細胞群叢形成促進因子(GM-CSF)處理細胞，syndecan-2和RACK1的作用不論是有無磷酸化的參與(phosphate-dependent or independent)皆有增強的現象，且有時間和劑量上的效應(time-and dose-dependent effect)，該現象推測可能是與syndecan-2的磷酸化及syndecan-2的叢聚現象(clustering)所造成，但仍需更進一步實驗證實。
最後，我們試著分析syndecan-2在細胞生理的功能，並探討與RACK1交互作用的相關性，來進一步了解這兩個分子交互作用可能的細胞生理意義。由上述實驗結果，我們知道syndecan-2胞內區域磷酸化會增加兩分子間的作用，因此我們建構了正常型(wild type：Syn-2-wt)、酪胺酸單點突變(Syn-2-Y180F、Syn-2-Y192F、Syn-2-Y180/192F)及胞內區域(intracellular domain：PEP Syn-2-cyto) 的syndecan-2表現載體，在細胞中進行表現後，分別以細胞型態、貼附、爬移和增生等生理現象和生化方式進行觀察和分析，我們發現在不同載體表現的轉染細胞中，其細胞生理現象的差異和syndecan-2 /RACK1磷酸化參與交互作用的部分具有關聯性，然而是否藉由該作用的差異性來調控可能的分子(如：Src kinase or Protein kinase c)進而造成細胞生理現象的差異，其中的分子機轉仍須進一步的實驗證明。在本實驗中雖然無法明確了解syndecan-2和RACK1交互作用的意義，但仍提供了將來可能的實驗方向。

In this study, RACK1 (Receptor for Activated C Kinase 1) was found to be reactive with syndecan-2 in vitro and in vivo. Through affinity column chromatography and immunoprecipitation analysis as well as immunocytochemical colocalization studies, the reaction between RACK1 and syndecan-2 was evidenced in BALB/3T3 cells. Recombinant syndecan-2 and PEP Syn-2-cyto were applied to demonstrate that tyrosine 180 of syndecan-2 is a targeted site for Src tyrosine kinase and the reaction with RACK1 is enhanced after this tyrosine phosphorylation. In parallel, when granulocyte-macrophage colony-stimulating factor (GM-CSF) was applied to activate cellular tyrosine kinase of HeLa cells, a significant positive interaction was revealed between syndecan-2 and RACK1 with time and dose-dependence.
HeLa cells were further subject to transfections with wild type and mutant syndecan-2 vectors(Syn-2-Y180F、Syn-2-Y192F、Syn-2-Y180/192F) to show that the reaction of syndecan-2 with RACK1 was suppressed when tyrosine 180 phosphorylation site was absent. To elucidate the physiological significance of this selective reaction between syndecan-2 and RACK1, studies with adhesion, migration, and poliferation were focused. HeLa cells transfected with Syn-2-Y180F mutant vectors exhibited less motile and adhesion ability. Furthermore, the morphology of Syn-2-Y180F transfants became round and refractile. These results imply that tyrosine 180 of syndecan-2 may involve in the cytoskeleton organization, focal contacts formation, and tyrosine kinase regulation through selective reaction with RACK1.

中文摘要..................................................1
ABSTRACT..................................................2
壹、前言
1.Syndecans 家族的特性...................................3
2.Syndecan-2的基本特性....................................4
3.RACK1的基本特性.........................................6
4.實驗動機................................................7
5.實驗架構................................................7
貳、器材與藥品
1. 生物材料...............................................8
2. 實驗材料...............................................8
3. 實驗設備...............................................8
4. 實驗藥品與試劑.........................................9
5. 引子的設計............................................14
參、實驗方法
抽取總量RNA..............................................15
置備cDNA.................................................15
聚合酶連鎖反應...........................................16
DNA 電泳和EtBr染色.......................................16
純化DNA產物..............................................17
syndecan-2 cDNA引子的設計與全長和胞內功能區域DNA片段的獲得.......................................................17
質體的構築...............................................18
構築小鼠全長與胞內功能區域syndecan-2 cDNA在細菌表現質體的
保存與定序...............................................19
小鼠全長與胞內功能區域syndecan-2 cDNA在細菌表現質體的構築19
構築全長與胞內功能區域syndecan-2 cDNA在哺乳動物細胞表現質
體的保存與序.............................................20
全長與胞內功能區域syndecan-2 cDNA在哺乳動物細胞表現質體的
構築.....................................................21
單鹼基點突變小鼠syndecan-2 cDNA 在細菌和哺乳動物細胞表現
質體的構築...............................................21
抽取小量質體.............................................23
細菌重組蛋白質的誘導表現.................................23
全長、胞內功能區域syndecan-2 和RACK1重組蛋白的分離與純化24
蛋白質定量...............................................25
Tricine-硫酸十二酯鈉聚丙烯醯氯膠電泳分析.................25
Coomassie blue R-250 染色法..............................26
西方點墨分析法...........................................26
細胞培養基...............................................26
細胞培養.................................................27
全長syndecan-2及其點突變株的細胞轉染.....................27
篩選轉染有全長syndecan-2及其點突變株的細胞株群...........28
細胞免疫染色.............................................28
細胞膜的粗萃.............................................28
免疫沉澱法...............................................29
細胞進行粒細胞/巨噬細胞群叢形成促進因子的處理............29
Hitrap-PEP taxol 和syndecan-2親和管柱的製備與操作........30
Hitrap- syndecan-2-PEP cyto 或RACK1 鎳離子嵌合親和管柱的
製備與作.................................................31
重組Src (p60c-src) 對syndecan-2、syndecan-2-Y180F
syndecan-2-PEP cyto的磷酸化作用..........................31
細胞增生分析.............................................32
細胞貼附分析.............................................32
細胞遷移能力分析.........................................33
肆、結果
全長和胞內syndecan-2 cDNA 的選殖以及點突變的建構和定序...34
小鼠全長和胞內syndecan-2重組蛋白的表現和純化.............34
以親和管柱色層分析證實重組蛋白RACK1和全長及胞內syndecan-2
具有交互作用的現象.......................................35
以親和管柱色層分析細胞中RACK1和syndecan-2-PEPCYTO重組蛋白
的交互作用，和免疫沉澱法證實syndecan-2和RACK1 complex 在
細胞中的存在.............................................36
以共軛焦顯微鏡觀察syndecan-2和RACK1在細胞中的重疊位置....36
syndecan-2-PEP cyto、syndecan-2和syndecan-2-Y180 F進行Src（p60 c-src）的磷酸化作用................................37
syndecan-2-PEP cyto在Src（p60 c-src）的磷酸化作用下有增強
與RACK1交互作用的現象....................................37
HeLa細胞在GM-CSF處理下，syndecan-2和RACK1交互作用有增強的
現象.....................................................38
HeLa細胞轉染syndecan-2基因作表現，觀察細胞型態...........39
HeLa細胞轉染syndecan-2及其突變株作基因表現後，分別進行細
胞貼附分析...............................................39
HeLa細胞轉染syndecan-2的突變株作基因表現後，觀察細胞型態
的差異...................................................40
HeLa細胞轉染syndecan-2及其突變株作基因表現後，分別進行細
胞遷移能力試驗...........................................41
HeLa細胞轉染syndecan-2及其突變株作基因表現後，進行細胞增
生分析...................................................42
HeLa細胞轉染syndecan-2胞內區域，觀察細胞型態的差異.......43
HeLa細胞轉染syndecan-2及其突變株作基因表現後，粗萃細胞膜
後分別進行免疫沉澱法.....................................43
伍、討論.................................................45
陸、參考文獻.............................................53
柒、圖表.................................................60

Angenstein, F., Evans, A. M., Settlage, R. E., Moran, S. T., Ling, S. C., Klintsova, A. Y., Shabanowitz, J., Hunt, D. F., & Greenough, W. T. (2002). A receptor for activated C kinase is part of messenger ribonucleoprotein complexes associated with polyA-mRNAs in neurons. J Neurosci, 22(20), 8827-8837.

Beauvais, D. M., & Rapraeger, A. C. (2004). Syndecans in tumor cell adhesion and signaling. Reprod Biol Endocrinol, 2, 3.

Besson, A., Wilson, T. L., & Yong, V. W. (2002). The anchoring protein RACK1 links protein kinase Cepsilon to integrin beta chains. Requirements for adhesion and motility. J Biol Chem, 277(24), 22073-22084.

Ceci, M., Gaviraghi, C., Gorrini, C., Sala, L. A., Offenhauser, N., Marchisio, P. C., & Biffo, S. (2003). Release of eIF6 (p27BBP) from the 60S subunit allows 80S ribosome assembly. Nature, 426(6966), 579-584.

Chang, B. Y., Harte, R. A., & Cartwright, C. A. (2002). RACK1: a novel substrate for the Src protein-tyrosine kinase. Oncogene, 21(50), 7619-7629.

Chen, L., Klass, C., & Woods, A. (2004). Syndecan-2 regulates transforming growth factor-beta signaling. J Biol Chem, 279(16), 15715-15718.

Choi, S., Lee, E., Kwon, S., Park, H., Yi, J. Y., Kim, S., Han, I. O., Yun, Y., & Oh, E. S. (2005). Transmembrane domain-induced oligomerization is crucial for the functions of syndecan-2 and syndecan-4. J Biol Chem, 280(52), 42573-42579.

Chong, Y. P., Ia, K. K., Mulhern, T. D., & Cheng, H. C. (2005). Endogenous and synthetic inhibitors of the Src-family protein tyrosine kinases. Biochim Biophys Acta, 1754(1-2), 210-220.

Chu, L. Y., Chen, Y. H., & Chuang, N. N. (2005). Dimerize RACK1 upon transformation with oncogenic ras. Biochem Biophys Res Commun, 330(2), 474-482.


Clasper, S., Vekemans, S., Fiore, M., Plebanski, M., Wordsworth, P., David, G., & Jackson, D. G. (1999). Inducible expression of the cell surface heparan sulfate proteoglycan syndecan-2 (fibroglycan) on human activated macrophages can regulate fibroblast growth factor action. J Biol Chem, 274(34), 24113-24123.

Contreras, H. R., Fabre, M., Granes, F., Casaroli-Marano, R., Rocamora, N., Herreros, A. G., Reina, M., & Vilaro, S. (2001). Syndecan-2 expression in colorectal cancer-derived HT-29 M6 epithelial cells induces a migratory phenotype. Biochem Biophys Res Commun, 286(4), 742-751.

David, G. (1992). Structural and functional diversity of the heparan sulfate proteoglycans. Adv Exp Med Biol, 313, 69-78.

Davies, E. J., Blackhall, F. H., Shanks, J. H., David, G., McGown, A. T., Swindell, R., Slade, R. J., Martin-Hirsch, P., Gallagher, J. T., & Jayson, G. C. (2004). Distribution and clinical significance of heparan sulfate proteoglycans in ovarian cancer. Clin Cancer Res, 10(15), 5178-5186.

Ethell, I. M., Hagihara, K., Miura, Y., Irie, F., & Yamaguchi, Y. (2000). Synbindin, A novel syndecan-2-binding protein in neuronal dendritic spines. J Cell Biol, 151(1), 53-68.

Ethell, I. M., Irie, F., Kalo, M. S., Couchman, J. R., Pasquale, E. B., & Yamaguchi, Y. (2001). EphB/syndecan-2 signaling in dendritic spine morphogenesis. Neuron, 31(6), 1001-1013.

Fears, C. Y., Gladson, C. L., & Woods, A. (2006). Syndecan-2 is expressed in the microvasculature of gliomas and regulates angiogenic processes in microvascular endothelial cells. J Biol Chem, 281(21), 14533-14536.

Gallay, P. (2004). Syndecans and HIV-1 pathogenesis. Microbes Infect, 6(6), 617-622.

Garcia-Higuera, I., Gaitatzes, C., Smith, T. F., & Neer, E. J. (1998). Folding a WD repeat propeller. Role of highly conserved aspartic acid residues in the G protein beta subunit and Sec13. J Biol Chem, 273(15), 9041-9049.



Geijsen, N., Spaargaren, M., Raaijmakers, J. A., Lammers, J. W., Koenderman, L., & Coffer, P. J. (1999). Association of RACK1 and PKCbeta with the common beta-chain of the IL-5/IL-3/GM-CSF receptor. Oncogene, 18(36), 5126-5130.

Identification of a novel Ezrin-binding site in syndecan-2 cytoplasmic domain. FEBS Lett, 547(1-3), 212-216.

Granes, F., Garcia, R., Casaroli-Marano, R. P., Castel, S., Rocamora, N., Reina, M., Urena, J. M., & Vilaro, S. (1999). Syndecan-2 induces filopodia by active cdc42Hs. Exp Cell Res, 248(2), 439-456.

Grootjans, J. J., Zimmermann, P., Reekmans, G., Smets, A., Degeest, G., Durr, J., & David, G. (1997). Syntenin, a PDZ protein that binds syndecan cytoplasmic domains. Proc Natl Acad Sci U S A, 94(25), 13683-13688.

Halden, Y., Rek, A., Atzenhofer, W., Szilak, L., Wabnig, A., & Kungl, A. J. (2004). Interleukin-8 binds to syndecan-2 on human endothelial cells. Biochem J, 377(Pt 2), 533-538.

Han, I., Park, H., & Oh, E. S. (2004). New insights into syndecan-2 expression and tumourigenic activity in colon carcinoma cells. J Mol Histol, 35(3), 319-326.

Huang, J. W., Chen, C. L., & Chuang, N. N. (2005). P120-GAP associated with syndecan-2 to function as an active switch signal for Src upon transformation with oncogenic ras. Biochem Biophys Res Commun, 329(3), 855-862.

Kim, Y., Park, H., Lim, Y., Han, I., Kwon, H. J., Woods, A., & Oh, E. S. (2003). Decreased syndecan-2 expression correlates with trichostatin-A induced-morphological changes and reduced tumorigenic activity in colon carcinoma cells. Oncogene, 22(6), 826-830.

Kinnunen, T., Kaksonen, M., Saarinen, J., Kalkkinen, N., Peng, H. B., & Rauvala, H. (1998). Cortactin-Src kinase signaling pathway is involved in N-syndecan-dependent neurite outgrowth. J Biol Chem, 273(17), 10702-10708.



Kiely, P. A.Leahy, M.O''Gorman, D.O''Connor, R.(2005). RACK1-mediated integration of adhesion and insulin-like growth factor I (IGF-I) signaling and cell migration are defective in cells expressing an IGF-I receptor mutated at tyrosines 1250 and 1251 J Biol Chem, 280(9), 7624-33

Klass, C. M., Couchman, J. R., & Woods, A. (2000). Control of extracellular matrix assembly by syndecan-2 proteoglycan. J Cell Sci, 113 ( Pt 3), 493-506.

Koehler, J. A., & Moran, M. F. (2001). RACK1, a protein kinase C scaffolding protein, interacts with the PH domain of p120GAP. Biochem Biophys Res Commun, 283(4), 888-895.

Kramer, K. L., Barnette, J. E., & Yost, H. J. (2002). PKCgamma regulates syndecan-2 inside-out signaling during xenopus left-right development. Cell, 111(7), 981-990.

Kusano, Y., Oguri, K., Nagayasu, Y., Munesue, S., Ishihara, M., Saiki, I., Yonekura, H., Yamamoto, H., & Okayama, M. (2000). Participation of syndecan 2 in the induction of stress fiber formation in cooperation with integrin alpha5beta1: structural characteristics of heparan sulfate chains with avidity to COOH-terminal heparin-binding domain of fibronectin. Exp Cell Res, 256(2), 434-444.

Liliental, J., & Chang, D. D. (1998). Rack1, a receptor for activated protein kinase C, interacts with integrin beta subunit. J Biol Chem, 273(4), 2379-2383.

Loreni, F., Iadevaia, V., Tino, E., Caldarola, S., & Amaldi, F. (2005). RACK1 mRNA translation is regulated via a rapamycin-sensitive pathway and coordinated with ribosomal protein synthesis. FEBS Lett, 579(25), 5517-5520.

Mamidipudi, V., Chang, B. Y., Harte, R. A., Lee, K. C., & Cartwright, C. A. (2004). RACK1 inhibits the serum- and anchorage-independent growth of v-Src transformed cells. FEBS Lett, 567(2-3), 321-326.

Midwood, K. S., Valenick, L. V., Hsia, H. C., & Schwarzbauer, J. E. (2004). Coregulation of fibronectin signaling and matrix contraction by tenascin-C and syndecan-4. Mol Biol Cell, 15(12), 5670-5677.

Modrowski, D., Basle, M., Lomri, A., & Marie, P. J. (2000). Syndecan-2 is involved in the mitogenic activity and signaling of granulocyte-macrophage colony-stimulating factor in osteoblasts. J Biol Chem, 275(13), 9178-9185.

Modrowski, D., Orosco, A., Thevenard, J., Fromigue, O., & Marie, P. J. (2005). Syndecan-2 overexpression induces osteosarcoma cell apoptosis: Implication of syndecan-2 cytoplasmic domain and JNK signaling. Bone, 37(2), 180-189.

Mourton, T., Hellberg, C. B., Burden-Gulley, S. M., Hinman, J., Rhee, A., & Brady-Kalnay, S. M. (2001). The PTPmu protein-tyrosine phosphatase binds and recruits the scaffolding protein RACK1 to cell-cell contacts. J Biol Chem, 276(18), 14896-14901.

Oh, E. S., Couchman, J. R., & Woods, A. (1997). Serine phosphorylation of syndecan-2 proteoglycan cytoplasmic domain. Arch Biochem Biophys, 344(1), 67-74.

Orosco, A., Fromigue, O., Hay, E., Marie, P. J., & Modrowski, D. (2006). Dual involvement of protein kinase C delta in apoptosis induced by syndecan-2 in osteoblasts. J Cell Biochem.

Park, H., Han, I., Kwon, H. J., & Oh, E. S. (2005). Focal adhesion kinase regulates syndecan-2-mediated tumorigenic activity of HT1080 fibrosarcoma cells. Cancer Res, 65(21), 9899-9905.

Park, H., Kim, Y., Lim, Y., Han, I., & Oh, E. S. (2002). Syndecan-2 mediates adhesion and proliferation of colon carcinoma cells. J Biol Chem, 277(33), 29730-29736.

Pass, J. M., Zheng, Y., Wead, W. B., Zhang, J., Li, R. C., Bolli, R., & Ping, P. (2001). PKCepsilon activation induces dichotomous cardiac phenotypes and modulates PKCepsilon-RACK interactions and RACK expression. Am J Physiol Heart Circ Physiol, 280(3), H946-955.

Quan, G. X., Krell, P. J., Arif, B. M., & Feng, Q. (2006). Receptor of activated C kinase 1 (RACK1) is necessary for the 20-hydroxyecdysone-induced expression of the transcription factor CHR3 in the spruce budworm Choristoneura fumiferana. Insect Mol Biol, 15(1), 79-87.
Reizes, O., Goldberger, O., Smith, A. C., Xu, Z., Bernfield, M., & Bickel, P. E. (2006). Insulin promotes shedding of syndecan ectodomains from 3T3-L1 adipocytes: a proposed mechanism for stabilization of extracellular lipoprotein lipase. Biochemistry, 45(18), 5703-5711.

Ron, D., Chen, C. H., Caldwell, J., Jamieson, L., Orr, E., & Mochly-Rosen, D. (1994). Cloning of an intracellular receptor for protein kinase C: a homolog of the beta subunit of G proteins. Proc Natl Acad Sci U S A, 91(3), 839-843.

Sang, N., Severino, A., Russo, P., Baldi, A., Giordano, A., Mileo, A. M., Paggi, M. G., & De Luca, A. (2001). RACK1 interacts with E1A and rescues E1A-induced yeast growth inhibition and mammalian cell apoptosis. J Biol Chem, 276(29), 27026-27033.

Schechtman, D., & Mochly-Rosen, D. (2001). Adaptor proteins in protein kinase C-mediated signal transduction. Oncogene, 20(44), 6339-6347.

Soldi, R., Primo, L., Brizzi, M. F., Sanavio, F., Aglietta, M., Polentarutti, N., Pegoraro, L., Mantovani, A., & Bussolino, F. (1997). Activation of JAK2 in human vascular endothelial cells by granulocyte-macrophage colony-stimulating factor. Blood, 89(3), 863-872.

Thornton, C., Tang, K. C., Phamluong, K., Luong, K., Vagts, A., Nikanjam, D., Yaka, R., & Ron, D. (2004). Spatial and temporal regulation of RACK1 function and N-methyl-D-aspartate receptor activity through WD40 motif-mediated dimerization. J Biol Chem, 279(30), 31357-31364.

Usacheva, A., Smith, R., Minshall, R., Baida, G., Seng, S., Croze, E., & Colamonici, O. (2001). The WD motif-containing protein receptor for activated protein kinase C (RACK1) is required for recruitment and activation of signal transducer and activator of transcription 1 through the type I interferon receptor. J Biol Chem, 276(25), 22948-22953.

Villena, J., Berndt, C., Granes, F., Reina, M., & Vilaro, S. (2003). Syndecan-2 expression enhances adhesion and proliferation of stably transfected Swiss 3T3 cells. Cell Biol Int, 27(12), 1005-1010.


Won, M., Park, S. K., Hoe, K. L., Jang, Y. J., Chung, K. S., Kim, D. U., Kim, H. B., & Yoo, H. S. (2001). Rkp1/Cpc2, a fission yeast RACK1 homolog, is involved in actin cytoskeleton organization through protein kinase C, Pck2, signaling. Biochem Biophys Res Commun, 282(1), 10-15.

Yamaguchi, Y., & Irie, F. (2002). [Roles of Eph receptors and the cell surface heparan sulfate proteoglycan syndecan-2 in dendritic spine morphogenesis]. Seikagaku, 74(5), 391-395.

Yarwood, S. J., Steele, M. R., Scotland, G., Houslay, M. D., & Bolger, G. B. (1999). The RACK1 signaling scaffold protein selectively interacts with the cAMP-specific phosphodiesterase PDE4D5 isoform. J Biol Chem, 274(21), 14909-14917.