肝醣合成激酶3 (Glycogen synthase kinase-3) 在許多訊息傳遞途徑中扮演著重要的角色。在大多數的真核細胞亦發現相似的肝醣合成激酶。因為肝醣合成激酶3在細胞中具有許多調控的功能，因此本研究利用酵母菌雙雜合系統 (yeast two-hybrid system) 由人類testis cDNA library進行肝醣合成激酶3β (GSK3β) 結合蛋白的篩選。在篩選出的GSK3β結合蛋白中，本論文選取兩個結合蛋白進一步來探討其功能，一是中心體蛋白質 hNinein，另一個則是新發現可抑制 GSK3β 活性的結合蛋白質 (GSK3β interaction protein; GSKIP)。
本論文第一部份研究發現中心體蛋白質 hNinein 具有四種不同C端區域 (CCII-termini) 的異構體 (isoforms)，其中hNinein isoform 6 為本研究的新發現；且 hNinein isoform 6 其分布情形具有組織特異性。GSK3β 對於四種不同C端區域的 isoforms 亦具有不同的磷酸化作用。此外切除 hNinein 的N端或 CCII 區域都會造成 hNinein 結構上的改變而影響其在細胞內的分布情形，進而造成另一個中心體蛋白質 γ-tubulin 的位置改變。進一步將細胞內所有 hNinein isoforms 移除後發現 γ -tubulin 在中心體的含量也相對減少。利用功能區域交換 (domain swapping) 的實驗，發現 CCIIX-CCIIY region 提供了一個 γ-tubulin 的結合位置。當細胞過度表達全長或 CCIIX-CCIIY region 的hNinein 蛋白質都會顯著影響微管束 (microtubules) 的生成。綜上所述，hNinein 具有中心體坐落標的訊號 (centrosomal targeting signals)，不僅可調控 hNinein 本身結構變化並提供與 γ-tubulin 的結合區域，進而影響其在細胞內分佈的情形。
本論文第二部份，就肝醣合成激酶3beta 的作用蛋白 (GSK3β interaction protein; GSKIP) 可抑制 GSK3β 對 non-primed 受質磷酸化的活性，利用神經母細胞瘤 (neuroblastoma SH-SY5Y) 當作神經分化的模式來進一步研究其功能。實驗發現當過度表達 GSKIP 可抑制 retinoic acid 誘發神經母細胞瘤 SH-SY5Y 神經軸 (neurite outgrowth) 的生長。此現象與利用 GSK3β 專一性抑制劑 LiCl 或 SB415286 進行相同實驗結果一致。進一步研究發現 GSKIP 可能藉由蛋白質交互作用 (protein-protein interactions) 而非其他蛋白質修飾作用(post-modulation) 來調控 GSK3β 的活性；且 GSKIP 可能藉由抑制 GSK3β 對 tau 蛋白質特定位點的磷酸化而影響神經軸的生長。此外經由細胞生長曲線及 MTT assay 分析發現 GSKIP 可促進細胞週期循環加速。同時 GSK3β的活性受 GSKIP 抑制進而造成細胞內 β-catenin 和 cyclin D1 總量增加。根據上述結果，GSKIP 在 retinoic acid 誘發神經母細胞瘤 SH-SY5Y 神經分化的模式中具有兩種調控功能，不僅抑制神經軸的生長亦促進細胞數的增生。而這兩種功能可能都是藉由對 GSK3β的活性的抑制所導致。

Glycogen synthase kinase-3 (GSK-3) is a serine/threonine protein kinase which plays a key role in several signaling pathways and its homologues have been identified in most eukaryotes. Since GSK3βis an essential protein kinase that regulates numerous functions within the cell, an effort to survey possible GSK3β- interacting proteins from a human testis cDNA library using the yeast two-hybrid system is made. Two interesting candidates are chosen to characterize their functions in this study. One is a centrosomal protein, hNinein, and the other is a novel inhibitor of GSK3β, designated as GSKIP (GSK3β interaction protein).
In the first part of the present thesis we describe the identification of four diverse CCII-termini of human hNinein isoforms, including a novel isoform 6, by differential expression in a tissue-specific manner. In a kinase assay, the CCII region of hNinein isoforms provides a differential phosphorylation site by GSK3β. In addition, either N-terminal or CCIIZ domain disruption may cause hNinein conformational change which recruits γ-tubulin to centrosomal or non-centrosomal hNinein-containing sites. Further, depletion of all hNinein isoforms caused a significant decrease in the γ-tubulin signal in the centrosome. In domain swapping, it clearly shows that the CCIIX-CCIIY region provides docking sites for γ-tubulin. Moreover, nucleation of microtubules from the centrosome is significantly affected by the overexpression of either the full-length hNinein or CCIIX-CCIIY region. Taken together, these results show that the centrosomal targeting signals of hNinein have a role not only in regulating hNinein conformation, resulting in localization change, but also provide docking sites to recruit γ-tubulin at centrosomal and non-centrosomal sites.
In the second part of the thesis we describe another candidate, GSK3βinteraction protein (GSKIP), to characterize its functions in neuron differentiation. We use human neuroblastoma SH-SY5Y cells as a model of neuronal cell differentiation. When overexpression of GSKIP prevents neurite outgrowth from RA-mediated differentiation, this result is similar to the presence of LiCl or SB415286, an inhibitor of GSK3β. Further, GSKIP regulates the activity of GSK3β through protein-protein interactions rather than post-modulation and GSKIP may affect GSK3β on neurite outgrowth via inhibiting the specific phosphorylation site of tau. In addition to inhibition of neurite outgrowth, GSKIP overexpressed in SH-SY5Y cells also promotes cell cycle progression by analyzing cell proliferation with cell growth and MTT assay. Furthermore, GSKIP raises the level of β-catenin and cyclin D1 through inhibition of GSK3β activity in RA-mediated differentiation SH-SY5Y cells. Taken together, the data suggest that GSKIP, a dual functional molecule, is able to inhibit neurite outgrowth and promote cell proliferation via negative regulation of GSK3β activity in RA-mediated differentiation of SH-SY5Y cells.

TABLE OF CONTENTS
TABLE OF CONTENTS I
ABSTRACT (Chinese) 1
ABSTRACT (English) 3
AN OVERVIEW OF THE GSK3 5
PART I: Functional Characterization of Human Ninein Isoforms – hNinein is
Regulated by Centrosomal Targeting Signals and Evidence for Docking
Sites to Direct γ-Tubulin 8
INTRODUCTION 9
MATERIALS AND METHODS 15
Isoform-specific PCR and RT-PCR 15
Cloning and DNA sequencing 15
Antibody production 16
Cell culture, RNA interference, and transfections 17
Immunofluorescence and microscopy 17
Western blot analysis 18
Immunoprecipitation and immunoblotting experiments 19
In vitro kinase assay 20
Microtubule regrowth experiment 20
RESULTS 21
Identification of four different human hNinein isoforms revealed a Novel hNinein
Isoform 21
Distribution of hNinein isoforms in the cell 22
C-terminal region of three hNinein isoforms may be regulated by GSK3β
phosphorylation 23
Both the N- and C-terminus contributed to hNinein conformation resulting in
γ-tubulin relocalization 23
Four targeting domains in hNinein are sufficient for centrosome localization
signals 25
hNinein is associated with γ-tubulin via CCIIX-CCIIY docking sites 26
Regrowth experiment with overexpression of full length and truncated hNinein 27
DISCUSSION 28
hNinein isoforms have different C-terminal coiled-coil domains 28
Redundant or distinct functions of hNinein isoforms 29
The distinct centrosomal localization of hNinein isoforms may be regulated by
GSK3β phosphorylation 30
γ-tubulin localization to the centosome is hNinein-dependent 30
Overexpression of hNinein inhibits microtubule nucleation and cell mitosis 32
hNinein isoforms are regulated by centrosome targeting signals and docks
γ-tubulin to the centosomal and non-centrosomal sites 33
PART II: GSKIP , a dual functional molecule, inhibites neurite outgrowth and
promotes cell proliferation via negative regulation of GSK3β activity
in retinoid acid-mediated differentiation of SH-SY5Y cells 35
INTRODUCTION 36
MATERIALS AND METHODS 39
Cell culture and Differentiation 39
Cloning and DNA Sequencing 39
Transfection 40
Immunofluorescence and microscopy 40
Western blotting 41
Cell growth cure and MTT assay 41
RESULTS 42
RA-mediated differentiation in human neuroblastoma SH-SY5Y cells 42
The inhibition of GSK3β prevents neurite outgrowth from RA-mediated
differentiation 42
GSKIP may affects GSK3β on neurite outgrowth via phosphorylated on specific
site of tau 43
GSKIP overexpression promotes cell cycle progression in SH-SY5Y cells 44
Overexpression of GSKIP raised the level of β-catenin and cyclin D1
REFERENCES 50
LIST OF FIGURES
Figure 1. Identification of a novel human hNinein isoform 65
Figure 2. The distribution of four hNinein isoforms in cells 68
Figure 3. Phosphorylation of four C-terminal fragments of hNinein isoforms by
GSK3β, Aurora A and PKA 72
Figure 4. C- and N-terminal domain disruptions of GFP-tagged hNinein isoform 5
were over-expressed in HeLa cells 73
Figure 5. Four centrosome localization signals in hNinein 76
Figure 6. hNinein is associated with γ-tubulin using the CCIIX-CCIIY docking
sites 79
Figure 7. Microtubules regrowth assay 82
Figure 8. Model for the structure and the role of hNinein in position, anchoring
and microtubule nucleation by the centrosome organization 85
Figure 9. Morphological changes in SH-SY5Y human neuroblastoma cells
differentiated by treatment with 10μM retinoic acid for 5 days 87
Figure 10. Inhibition of GSK3β activity blocks neurite outgrowth in SH-SY5Y
human neuroblastoma cells 88
Figure 11. Overexpression of GSKIP inhibits neurite outgrowth in SH-SY5Y
human neuroblastoma cells 90
Figure 12. GSKIP has no effects on phosphorylation of GSK3β 91
Figure 13. GSKIP affects GSK3β to phosphorylate tau on specific site 92
Figure 14. GSKIP overexpression promotes cell cycle progression in SH-SY5Y
cells 93
Figure 15. Overexpression of GSKIP increase the level of β-catenin and cyclin D1 94
Figure 16. Overexpression of GSKIP results in cyclin D1 acumination in nucleus 95
LIST OF TABLES
Table 1. Summary of subcellular localization, intrinsic targeting signals, γ-tubulin
docking site and phosphorylation site of various hNinein isoforms 96
Table 2. Summary of different hNinein portions when overexpressed in transfected
cells. 97
Table 3. Summary of GSK3β inhibitors affect on neurite outgrowth in RA-mediated
differentiation SH-SY5Y cells 98
APPENDIX I The centrosome structure 99
APPENDIX II The Centrosome Duplication Cycle 100
APPENDIX III ABBREVIATIONS 101
APPENDIX IV Antibody for immunofluorescenc 102
APPENDIX V 103

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