臺灣博碩士論文加值系統

Receptor for Activated C-Kinase 1 (RACK1)是一個分子量為36 kDa的鷹架蛋白，普遍存在於動物和植物中，RACK1的蛋白質結構是由七個平板螺旋 (seven-bladed propeller) 所組成，其中包含七個色胺基酸和天冬胺酸 (tryptophan-aspartic acid-domain；WD40) 的重複區域，而這些重複的區域 (domain) 也是與其他蛋白發生交互作用的地方。在阿拉伯芥中，有三個基因可以轉譯出RACK1蛋白，分別命名為RACK1A (At1g18080), RACK1B (At1g48630) 和RACK1C (At3g18130)。阿拉伯芥中的RACK1蛋白會參與在賀爾蒙反應和植物生長發育的過程中，且會與40S核醣體發生交互作用。目前，RACK1被視為一多功能性的鷹架蛋白，在不同的訊息傳遞途徑中扮演重要的角色。此外，RACK1在離層酸 (abscisic acid; ABA) 反應中扮演一個負向調控的角色，而rack1a突變株對於水分逆境具有抗性。然而，RACK1A究竟是與哪些蛋白質發生交互作用，共同參與或調控這些訊息傳遞的過程，都是尚未清楚的部分。前人研究以14-3-3抗體進行免疫沉澱 (immunoprecipitation) 的實驗，發現到RACK1A的存在。於是，我們想要再進一步去確認14-3-3蛋白與RACK1A蛋白之間的交互作用。由酵母菌雙雜合系統和雙分子螢光互補實驗得知，14-3-3蛋白與RACK1A蛋白之間沒有直接的交互作用。此外，觀察阿拉伯芥RACK1A在原生質體細胞內主要分布在細胞質，C端某絲胺酸定點突變導致RACK1A分布集中在核中。本研究並利用RACK1A大量表現植株，試著將RACK1A及其結合蛋白利用免疫沉澱方式純化出來。

Receptor for Activated C-Kinase 1 (RACK1) is a 36-kDa scaffold protein, which contains seven Trp-Asp 40 (WD40) repeats. RACK1 is highly conserved protein in both animals and plants. The WD40 repeats are involved in protein-protein interactions. There are three RACK1 genes in Arabidopsis thaliana, namely RACK1A (At1g18080), RACK1B (At1g48630) and RACK1C (At3g18130). RACK1 is involved in multiple hormone responses, developmental processes, and associated with 40S ribosomes in Arabidopsis. Notably, RACK1 is regarded as a versatile scaffold protein which serves as a nexus for multiple signal transduction pathways. Moreover, RACK1 plays a negative regulator under abscisic acid (ABA) responses, and rack1a mutants are more resistant to water stress. However, the specific interactions between RACK1 and its binding partners are still unclear. Previous immunopreciptation studies revealed that RACK1A may interact with 14-3-3. Here, we used yeast two-hybrid and bimolecular fluorescence complementation to study the interactions between these two proteins. Our results indicated that RACK1A did not interact with 14-3-3 omega directly. Furthermore, we observed that the RACK1A protein is localized to the cytoplasm in Arabidopsis protoplasts. Point mutation of a serine residue located in the C-terminue resulted in subcellular localization change. We use RACK1A overexpression lines to the possibility to identify RACK1A interacting proteins by immunoprecipitation.

目錄
中文摘要 V
Abstract VI
縮寫對照表 VII
第一章　前言 1
一、RACK1 (Receptor for Activated C-Kinase 1) 蛋白 1
1. RACK1的起源 1
2. RACK1的蛋白結構 1
3. RACK1的生理功能 2
二、14-3-3蛋白 3
三、RACK1蛋白與14-3-3蛋白的關係 4
四、研究目標 4
第二章　材料與方法 5
一、實驗材料 5
1. 質粒 (plasmid) 5
2. 菌株 5
二、實驗方法 5
1. 大腸桿菌勝任細胞 (competent cell) 之製備 5
2. 少量質粒DNA之抽取 6
3. DNA洋菜膠體電泳 7
4. 細菌轉型作用 (tansformation) 7
5. 榖胱甘肽硫轉移酶 (glutathione-S-transferase ; GST) 重組蛋白之純化 7
6. 蛋白質定量 8
7. 變性聚丙烯胺膠體電泳 (SDS-PAGE) 9
8. 西方墨點法 (western blot) 10
9. 阿拉伯芥原生質體 (protoplast) 的分離和質粒轉殖 10
10. 酵母菌雙雜合系統 (yeast two-hybrid system) 11
11. 重組蛋白之定點突變 (site-directed mutagenesis) 13
12. 試管內蛋白質磷酸化之激酶反應 (in vitro kinase assay) 14
13. 免疫沉澱 (immunoprecipitation) 14
14. 植物全蛋白之抽取及免疫沉澱方法 15
第三章　結果 16
一、以酵母菌雙雜合系統分析阿拉伯芥RACK1A與14-3-3 omega之分子間交互作用 16
二、阿拉伯芥RACK1A受激酶磷酸化之分析 17
三、以雙分子螢光互補系統 (BiFC) 分析阿拉伯芥RACK1A和14-3-3 omega之交互作用 18
四、觀察阿拉伯芥RACK1A在阿拉伯芥原生質體細胞內的分布 18
五、阿拉伯芥RACK1A大量表現株中HF-RAK1A的表現量 19
六、以GST-RACK1A融合蛋白進行免疫沉澱 19
七、以阿拉伯芥RACK1A大量表現的轉殖株進行免疫沉澱 19
第四章　討論 21
參考文獻 23
附錄 35

圖表目錄
圖一、RACK1A蛋白質序列及預期會與14-3-3蛋白發生交互作用的磷酸化位點 26
圖二、以酵母菌雙雜合系統分析阿拉伯芥14-3-3 omega和RACK1A之交互作用 27
圖三、GST-RACK1A重組蛋白 (fusion protein) 純化後的品質和濃度檢測 28
圖四、阿拉伯芥RACK1A分別與AtCDPK1、AtCDPK16和AtCRK3進行激酶活性分析 29
圖五、以雙分子螢光互補系統 (BiFC) 分析阿拉伯芥14-3-3 omega蛋白和RACK1A蛋白之交互作用 30
圖六、觀察YFP-RACK1A和YFP-RACK1A-S286A在阿拉伯芥原生質體中細胞內的位置 31
圖七、以西方墨點法分析阿拉伯芥RACK1A大量表現株中的RACK1A蛋白的表現量 32
圖八、利用GST-RACK1A重組蛋白進行免疫沉澱 (immunoprecipitation) 分析 33
圖九、以anti-RACK1 antibody進行免疫沉澱 (immunoprecipitation) 分析 34

Bachmann, M., Shiraishi, N., Campbell, W.H., Yoo, B.C., Harmon, A.C., and Huber, S.C. (1996). Identification of Ser-543 as the major regulatory phosphorylation site in spinach leaf nitrate reductase. Plant Cell 8: 505-517.
Bradford, M.M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248-254.
Chang, I.F., Szick-Miranda, K., Pan, S., and Bailey-Serres, J. (2005). Proteomic characterization of evolutionarily conserved and variable proteins of Arabidopsis cytosolic ribosomes. Plant Physiol 137: 848-862.
Chang, I.F., Curran, A., Woolsey, R., Quilici, D., Cushman, J.C., Mittler, R., Harmon, A., and Harper, J.F. (2009). Proteomic profiling of tandem affinity purified 14-3-3 protein complexes in Arabidopsis thaliana. Proteomics 9: 2967-2985.
Chen, J.G., Guo, J.J., Wang, S.C., Wang, J.B., Huang, W.D., and Liang, J.S. (2009). Dissection of the Relationship Between RACK1 and Heterotrimeric G-Proteins in Arabidopsis. Plant and Cell Physiology 50: 1681-1694.
Chen, J.G., Ullah, H., Temple, B., Liang, J., Guo, J., Alonso, J.M., Ecker, J.R., and Jones, A.M. (2006). RACK1 mediates multiple hormone responsiveness and developmental processes in Arabidopsis. J Exp Bot 57: 2697-2708.
Chen, S.H., Lin, F., and Hamm, H.E. (2005). RACK1 binds to a signal transfer region of G beta gamma and inhibits phospholipase C beta 2 activation. J Biol Chem 280: 33445-33452.
Chen, S.H., Dell, E.J., Lin, F., and Hamm, H.E. (2004). RACK1 regulates specific functions of G beta gamma. Faseb J 18: A973-A973.
DeLille, J.M., Sehnke, P.C., and Ferl, R.J. (2001). The arabidopsis 14-3-3 family of signaling regulators. Plant Physiol 126: 35-38.
Dell, E.J., Connor, J., Chen, S.H., Stebbins, E.G., Skiba, N.P., Mochly-Rosen, D., and Hamm, H.E. (2002). The beta gamma subunit of Heterotrimeric G proteins interacts with RACK1 and two other WD repeat proteins. J Biol Chem 277: 49888-49895.
Douglas, P., Moorhead, G., Hong, Y., Morrice, N., and MacKintosh, C. (1998). Purification of a nitrate reductase kinase from Spinacea oleracea leaves, and its identification as a calmodulin-domain protein kinase. Planta 206: 435-442.
Ferl, R.J., and Manak, M.S. (2007). Divalent cation effects on interactions between multiple Arabidopsis 14-3-3 isoforms and phosphopeptide targets. Biochemistry-Us 46: 1055-1063.
Giavalisco, P., Wilson, D., Kreitler, T., Lehrach, H., Klose, J., Gobom, J., and Fucini, P. (2005). High heterogeneity within the ribosomal proteins of the Arabidopsis thaliana 80S ribosome. Plant Mol Biol 57: 577-591.
Guo, J., and Chen, J.G. (2008). RACK1 genes regulate plant development with unequal genetic redundancy in Arabidopsis. BMC Plant Biol 8: 108.
Guo, J., Wang, J.B., Xi, L., Huang, W.D., Liang, J.S., and Chen, J.G. (2009). RACK1 is a negative regulator of ABA responses in Arabidopsis. Journal of Experimental Botany 60: 3819-3833.
Guo, J., Wang, S.C., Valerius, O., Hall, H., Zeng, Q.N., Li, J.F., Weston, D.J., Ellis, B.E., and Chen, J.G. (2011). Involvement of Arabidopsis RACK1 in Protein Translation and Its Regulation by Abscisic Acid. Plant Physiology 155: 370-383.
Huang, J.Z., Hardin, S.C., and Huber, S.C. (2001). Identification of a novel phosphorylation motif for CDPKs: phosphorylation of synthetic peptides lacking basic residues at P-3/P-4. Arch Biochem Biophys 393: 61-66.
Igarashi, D., Ishida, S., Fukazawa, J., and Takahashi, Y. (2001). 14-3-3 proteins regulate intracellular localization of the bZIP transcriptional activator RSG. Plant Cell 13: 2483-2497.
Ishida, S., Takahashi, Y., and Nagata, T. (1993). Isolation of Cdna of an Auxin-Regulated Gene Encoding a G-Protein Beta-Subunit-Like Protein from Tobacco by-2-Cells. P Natl Acad Sci USA 90: 11152-11156.
Ishida, S., Yuasa, T., Nakata, M., and Takahashi, Y. (2008). A tobacco calcium-dependent protein kinase, CDPK1, regulates the transcription factor REPRESSION OF SHOOT GROWTH in response to gibberellins. Plant Cell 20: 3273-3288.
Jones, D.H., Ley, S., and Aitken, A. (1995). Isoforms of 14-3-3-Protein Can Form Homodimers and Heterodimers in-Vivo and in-Vitro - Implications for Function as Adapter Proteins. Febs Lett 368: 55-58.
Kaiser, W.M., and Huber, S.C. (2001). Post-translational regulation of nitrate reductase: mechanism, physiological relevance and environmental triggers. J Exp Bot 52: 1981-1989.
Ke, Y., Han, G., He, H., and Li, J. (2009). Differential regulation of proteins and phosphoproteins in rice under drought stress. Biochem Biophys Res Commun 379: 133-138.
Komatsu, S., Abbasi, F., Kobori, E., Fujisawa, Y., Kato, H., and Iwasaki, Y. (2005). Proteomic analysis of rice embryo: an approach for investigating Galpha protein-regulated proteins. Proteomics 5: 3932-3941.
Laemmli, U.K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685.
McCahill, A., Warwicker, J., Bolger, G.B., Houslay, M.D., and Yarwood, S.J. (2002). The RACK1 scaffold protein: a dynamic cog in cell response mechanisms. Mol Pharmacol 62: 1261-1273.
McKhann, H.I., Frugier, F., Prteovics, G., de la Pe˜na, T., Jurkevitch, E., Brown, S., Kondorosi, E., Kondorosi, A., and Crespi, M. (1997). Cloning of aWD-repeat-containing gene from alfalfa (Medicago sativa): a role in hormone-mediated cell division? Plant Mol Biol 34: 771-780.
Mochly-Rosen, D., Khaner, H., and Lopez, J. (1991). Identification of intracellular receptor proteins for activated protein kinase C. Proc Natl Acad Sci U S A 88: 3997-4000.
Nakashima, A., Chen, L., Thao, N.P., Fujiwara, M., Wong, H.L., Kuwano, M., Umemura, K., Shirasu, K., Kawasaki, T., and Shimamoto, K. (2008). RACK1 functions in rice innate immunity by interacting with the Rac1 immune complex. Plant Cell 20: 2265-2279.
Paul, A.L., Liu, L., McClung, S., Laughner, B., Chen, S., and Ferl, R.J. (2009). Comparative Interactomics: Analysis of Arabidopsis 14-3-3 Complexes Reveals Highly Conserved 14-3-3 Interactions between Humans and Plants. J Proteome Res 8: 1913-1924.
Ron, D., Chen, C.H., Caldwell, J., Jamieson, L., Orr, E., and 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: 839-843.
Sehnke, P.C., DeLille, J.M., and Ferl, R.J. (2002). Consummating signal transduction: the role of 14-3-3 proteins in the completion of signal-induced transitions in protein activity. Plant Cell 14 Suppl: S339-354.
Sklan, E.H., Podoly, E., and Soreq, H. (2006). RACK1 has the nerve to act: structure meets function in the nervous system. Prog Neurobiol 78: 117-134.
Ullah, H., Scappini, E.L., Moon, A.F., Williams, L.V., Armstrong, D.L., and Pedersen, L.C. (2008). Structure of a signal transduction regulator, RACK1, from Arabidopsis thaliana. Protein Sci 17: 1771-1780.
Yoo, S.D., Cho, Y.H., and Sheen, J. (2007). Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nat Protoc 2: 1565-1572.