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研究生： 李怡萱
Li, Yi-Hsuan
論文名稱： 訊息蛋白SH2B1β藉由影響細胞黏著調控神經生長因子在PC12細胞誘發之神經軸突生長
The Adaptor Protein SH2B1β Regulates NGF-induced Neurite Outgrowth in PC12 Cells Through Modulating Cell Adhesion
指導教授： 陳令儀
Linyi Chen
口試委員:
學位類別： 碩士
Master
系所名稱： 生命科學暨醫學院 - 分子醫學研究所
Institute of Molecular Medicine
論文出版年： 2009
畢業學年度： 97
語文別： 英文
論文頁數： 73
中文關鍵詞： 神經生長因子 、細胞黏著 、神經軸突生長 、SH2B1beta 、PC12 細胞
外文關鍵詞： SH2B1beta, NGF, PC12 cells, cell adhesion, neurite outgrowth
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    • Neuronal precursor cells are differentiated into functional neurons during embryonic development. Various processes regulate neuronal differentiation, including signal transduction, gene expression, and cell adhesion. PC12 cell line is a well-established model for studying neuronal differentiation. After nerve growth factor (NGF) treatment, activation of the receptor tropomyosin-related kinase A (TrkA) induces downstream signaling such as mitogen-activated protein kinases (MAPK) cascade, phosphatidylinositol 3 kinase (PI3K)-Akt, phospholipase C gamma (PLCγ) and ultimately leads to neurite outgrowth in PC12 cells. Our previous results have shown that SH2B1β-overexpressing PC12 cells (PC12-SH2B1β cells) enhance neurite outgrowth compared with control PC12 cells (PC12-GFP cells). On the other hand, cells overexpressing the dominant negative form of SH2B1β [PC12-SH2B1β(R555E) cells] significantly reduce NGF-induced neuronal differentiation. Nevertheless, the effect of SH2B1β on NGF-induced neurite outgrowth is solely on MAPK, PI3K-Akt and PLCγ signaling because PC12-SH2B1β(R555E) cells do not inhibit NGF initiated signaling pathways. Interestingly, we have found that PC12-SH2B1β(R555E) cells show much more aggregated phenotype than PC12-SH2B1β and PC12-GFP cells. This result reveals the possibility that SH2B1β may regulate NGF-induced neuronal differentiation by modulating cell adhesion. In line with this finding, our preliminary results suggest a subset of NGF responsive genes including N-cadherin, which participates in cell-cell adhesion, is differentially regulated by SH2B1β and SH2B1β(R555E). This thesis investigated the possible mechanisms by which SH2B1β and SH2B1β(R555E) would regulate NGF-induced neurite outgrowth. The data in this thesis suggest that overexpressing SH2B1β□reduces the expression of N-cadherin and its binding to β-catenin. Moreover, overexpressing SH2B1β□also□increases the phosphorylation of β-catenin on Y654, and disrupts the association between N-cadherin and β-catenin. Furthermore, in response to NGF, SH2B1β□stabilizes the interaction between N-cadherin and β-catenin likely through modulating cyclin-dependent kinase 5 (CDK5) and Src kinase and thus downregulating the level of pβ-catenin (Y654). In contrast, in the absence of NGF, overexpressing SH2B1β(R555E) increases the expression of N-cadherin and its binding to β-catenin through inhibiting the phosphorylation of β-catenin on Y654. Taken together, the data in this thesis suggest that overexpressing SH2B1β downregulates the N-cadherin-β-catenin complex in the absence of NGF to allow faster neurite initiation, but increases the complex formation in response to NGF likely to facilitate cell-cell communication during neuronal differentiation. On the contrary, overexpressing SH2B1β(R555E) increases the interaction between N-cadherin and β-catenin and ultimately blocks neurite outgrowth.

    謝誌...............................................I Abstract...........................................II 中文摘要...........................................IV Index..............................................VI Abbreviations......................................VIII Introduction.......................................1 Materials and Methods..............................12 Reagents...........................................12 Cell culture and drug treatment....................14 Western Blotting...................................15 Co-immunoprecipitation.............................16 Immunostaining.....................................18 Total RNA extraction...............................18 Reverse transcription and semi-quantitative real time polymerase chain reaction..........................19 Statistical analysis...............................20 Results............................................22 SH2B1β and SH2B1β(R555E) differentially regulate the expression of N-cadherin...........................22 SH2B1β and SH2B1β(R555E) differentially affect NGF-induced β-catenin levels and the interaction between β-catenin and N-cadherin.........................................23 SH2B1β is not in the N-cadherin-β-catenin complex..27 ADAM10 probably does not play a major role in regulating N-cadherin stability in PC12-SH2B1β cells............27 Src and CDK5 regulate the interaction between N-cadherin and β-catenin......................................28 The working model for the role of SH2B1β and SH2B1β(R555E) in regulating N-cadherin-β-catenin complex during NGF-induced neurite outgrowth in PC12 cells............31 Discussion.........................................33 References.........................................38 Figures............................................42 Fig.1 SH2B1β and SH2B1β(R555E) differentially regulate the expression of N-cadherin...........................42 Fig.2 SH2B1β and SH2B1β(R555E) differentially regulate the expression of β-catenin............................44 Fig.3 The cellular localization of N-cadherin and β-catenin among PC12-GFP, PC12-SH2B1β□and PC12-SH2B1β(R555E) cells......................................46 Fig.4 SH2B1β enhances the association between N-cadherin and β-catenin in response to NGF...................48 Fig.5 The expression of ADAM10 in PC12-GFP, PC12-SH2B1β and PC12-SH2B1β(R555E) cells.......................50 Fig.6 Src does not bind to N-cadherin in PC12 cells.51 Fig.7 SH2B1β□and SH2B1β(R555E) regulate the binding of β-catenin to the CDK5-p35 complex....................52 Fig.8 Role of CDK5 and Src in the phosphorylation of β-catenin on Y654....................................55 Fig.9 CDK5 and Src regulate the binding between N-cadherin and β-catenin......................................56 Fig.10 The working model for the role of SH2B1β□and SH2B1β(R555E) in regulating N-cadherin-β-catenin complex during NGF-induced neuronal differentiation in PC12 cells.58 Tables.............................................59 Table 1. Reverse transcription protocol used in this thesis.............................................59 Table 2. Reaction temperature and time of reverse transcription......................................60 Table 3. Sequences of the Q-PCR primers used in this thesis.............................................61 Table 4. Reaction temperature and time of Q-PCR....62 Appendix...........................................63 Fig. A1 The cellular localization of β-catenin in PC12-SH2B1β□cells distributes in both the cytoplasm and the nuclei after LiCl treatment........................63 Fig. A2 The induced fold of luciferase activity after LiCl treatment is the strongest in PC12-SH2B1β□cells...65 Fig. A3 The cellular localization of pβ-catenin(Y654) in PC12-GFP, PC12-SH2B1β□and PC12-SH2B1β(R555E) cells.67 Fig. A4 The nuclear fraction of CTF2 in the PC12-GFP, PC12-SH2B1β□and PC12-SH2B1β(R555E) cells ...............68 Fig. A5 The expression of L1cam is reduced by overexpressing SH2B1β but increased by overexpressing SH2B1β(R555E)...........................................70 References for Appendix............................73

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