肝醣合成酶激酶-3beta (GSK3beta)是一個參與細胞內多元化調控過程的主要酵素，包括細胞發育、代謝平衡、神經生長及分化、細胞極性、細胞增殖與凋亡。過去數年間，我們實驗室致力於利用酵母菌雙雜交系統鑑定出多個GSK3beta的特殊結合蛋白，包括dynamin-like protein (Drp1)、 hNinein、 CGI-99、 CABYR variant、 GSKIP 以及 Astrin。近來，我們也利用此一平台篩選出一個未知的Aurora及hNinein的結合蛋白，AIBp。本篇論文的第一個部分，我們延續對Drp1的研究。Drp1是粒線體分裂蛋白，可調控粒線體形態，並與神經退化疾病有關。我們清楚地闡述了DRP1 634至736胺基酸的區域以及 Lys 679的位置是與GSK3beta結合所必需的。GSK3beta會磷酸化DRP1 Ser693的位置去促進粒線體型態的延長，降低cytochrome c 的釋放以及capase-3、caspase-7、PARP的活化因而可以抵抗過氧化氫所造成的細胞凋亡。論文的第二部分則發現AIBp也會與Plk1結合，因而促進Aurora A對Plk1的活化。相反地，AIBp卻會阻斷Ninein被Aurora A及Plk1的磷酸化作用。當降低AIBp表現量時，則會減少Aurora A 在Thr288, 以及Plk1 Thr210位置上的磷酸化，還有 ch-TOG在中心體上的錯誤定位，造成多個spindle pole的形成以及染色體的排列錯誤。 因此，我們推測AIBp與hNinein、Aurora A、Plk1之間的相互作用可能會促進細胞進入分裂期與調控紡錘絲的形成。

Glycogen synthase kinase-3beta (GSK3beta) has been reported to participate in several signaling pathways and crucial for cell development, metabolic homeostasis, neuronal growth and differentiation, cell polarity, cell fate and apoptosis. In the past several years, we pay great efforts to study protein-protein interactions by yeast two-hybrid techniques, and identified many novel GSK3beta interaction proteins including dynamin-like protein (Drp1), hNinein, CGI-99, CABYR variant, GSKIP and Astrin. Recently, we also used two-hybrid platform to discover a novel Aurora A and hNinein binding protein, AIBp. At first part of this study, we continued our research on Drp1. Drp1 is a mitochondria fission protein, involved in neurodegenerative diseases in terms of affecting mitochondrial dynamics. We clearly demonstrated that GSK3beta-mediated phosphorylation at Ser693 of Drp1 and the region of Drp1634-690 and Lys679 are crucial for GSK3beta interaction. We also revealed that mitochondrial elongation due to ectopic expression of Drp1 S693D mutants may be associated with enhanced resistance to H2O2-induced mitochondrial fragmentation and ensuing apoptosis via down-regulating cytochrome c release, capase-3, -7 and PARP activation, rather than inducing autophagy. At the second part of the study, we demonstrated that AIBp also interacts with Plk1, which subsequently promotes Aurora-A–mediated Plk1 activation. In contrast, it blocks the hNinein phosphorylation mediated by Aurora A and Plk1. Knockdown of AIBp expression caused down-regulation of Aurora A Thr288, Plk1 Thr210 phosphorylation and mislocalization of ch-TOG to centrosomes, resulted in phenotypes of multiple spindle pole formation and chromosome misalignment. We suggested that the interplay of hNinein, AIBp, Aurora-A, and Plk1 may contributes to mitotic entry and bipolar spindle assembly during mitotic progression.

論文審定書．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．ⅰ
誌謝(ACKNOWLEDGEMENTS) ．．．．．．．．．．．．．．．．．．．．．．．．．．ⅱ
中文摘要．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．ⅲ
ABSTRACT (English)．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．ⅳ
INDEX．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．ⅵ
LIST of FIGURES．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．ⅷ OVERVIEW．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．1
PART I: GSK3beta-mediated Drp1 phosphorylation induced elongated mitochondrial
morphology against oxidative stress ．．．．．．．．．．．．．．．．．．．．．5
SUMMARY．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．6
INTRODUCTION．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．． 8
1. Glycogen synthase kinase-3．．．．．．．．．．．．．．．．．．．．．．．．．． 8
2. Regulations of GSK3 activity．．．．．．．．．．．．．．．．．．．．．．．．． 8
3. Dynamin related protein 1．．．．．．．．．．．．．．．．．．．．．．．．．．． 10
4. Post-translational modifications of Drp1．．．．．．．．．．．．．．．．．．．．． 12
MATERIALS and METHODS．．．．．．．．．．．．．．．．．．．．．．．．．．．16
1. Cell culture and transfection．．．．．．．．．．．．．．．．．．．．．．．．．．16
2. Cloning, site-directed mutagenesis and DNA sequencing．．．．．．．．．．．．．． 17
3. Yeast two hybrid system．．．．．．．．．．．．．．．．．．．．．．．．．．． 18
4. Western blot analysis．．．．．．．．．．．．．．．．．．．．．．．．．．．．．18
5. In vitro kinase assay．．．．．．．．．．．．．．．．．．．．．．．．．．．．． 19
6. GTPase activity assay．．．．．．．．．．．．．．．．．．．．．．．．．．．．．20
7. Immunofluorescence and quantification of mitochondrial morphology．．．．．．．．． 20
8. Statistics．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．21
RESULTS．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．22
1. Interaction and co-localization of Drp1 with GSK3beta．．．．．．．．．．．．．．．．22
2. Drp1 Ser693 is a GSK3beta phosphorylation site．．．．．．．．．．．．．．．．．．．23
3. Drp1 S693 and K679 locate in GED and do not affect inter-/intra- molecular interactions, but K679 determines GSK3beta binding．．．．．．．．．．．．．．．．．．．．．．．．25
4. Drp1 Ser693 is involved in regulating its GTPase activity．．．．．．．．．．．．．．．27
5. Phosphorylation of Drp1 at Ser693 inhibits mitochondrial fission．．．．．．．．．．．．28
6. Overexpressed Drp1 S693D leads to resistance to H2O2-induced mitochondrial fragmentation and ensuing apoptosis, but did not induce autophagy．．．．．．．．．．．．．．．．．30
DISCUSSION．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．32
PART II: AIBp regulates mitotic entry and mitotic spindle assembly by controlling activation of both Aurora-A and Plk1．．．．．．．．．．．．．．．．．．．．．．．．．．．．．63
SUMMARY．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．64
INTRODUCTION．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．65
1. Centrosome．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．65
2. Aurora kinase A．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．66
3. Bora．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．67
4. Polo-like kinase 1．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．68
5. Ninein．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．70
6. AIBp．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．71
MATERIALs and METHODS．．．．．．．．．．．．．．．．．．．．．．．．．．．．75
1. Plasmid construction．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．75
2. Yeast two-hybrid system．．．．．．．．．．．．．．．．．．．．．．．．．．．．．75
3. Protein purification．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．76
4. Site-directed mutagenesis．．．．．．．．．．．．．．．．．．．．．．．．．．．．77
5. In vitro kinase assay．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．78
6. Cell culture, synchronization, RNA interference and transfection．．．．．．．．．．．．78
7. Immunofluorescence．．．．．．．．．．．．．．．．．．．．．．．．．．．．．79
8. Western blot analysis．．．．．．．．．．．．．．．．．．．．．．．．．．．．．80
9. Co-immunoprecipitation．．．．．．．．．．．．．．．．．．．．．．．．．．．．81
RESULTS．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．83
1. AIBp bridges hNinein and Aurora-A and anchors PLK1 to form a complex．．．．．．．．83
2. AIBp, Aurora-A and Plk1 co-localized to the spindle poles during early mitosis．．．．．．．83
3. AIBp acts as both a substrate and a positive regulator of Aurora-A and Plk1．．．．．．．．84
4. AIBp promotes Aurora-A–mediated Plk1 activation．．．．．．．．．．．．．．．．．．85
5. Knockdown of hNinein blocks centrosomal targeting of AIBp, whereas depletion of AIBp do not affect the localization of hNinein and microtubule nucleation．．．．．．．．．．．．．．86
6. AIBp depletion decrease phosphorylation of Plk1 Thr210 and Aurora-A Thr288．．．．．．87
7. Depletion of AIBp causes asymmetrical spindle poles, multipolar spindles, donut-shaped chromosomes, and chromosome misalignment．．．．．．．．．．．．．．．．．．．．88
8. AIBp was linked to mislocalization of TACC3 and ch-TOG to centrosomes．．．．．．．．89
DISCUSSION．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．91
1. AIBp acts as a positive and negative regulator towards both Aurora-A and Plk1．．．．．．．91
2. The function of AIBp is similar to that of Bora, but its role is to facilitate by interacting with hNinein and is involved in regulation of centrosomal microtubule signaling．．．．．．．．92
3. Formation of multiple spindle poles．．．．．．．．．．．．．．．．．．．．．．．．93

REFERENCES．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．116
APPENDIX ．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．136

List of figures

Figure ⅰ. The GSK3 and its partners involve in multiple biophysiological and pathophysiological activities. ．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．4
Figure1-1. Schematic illustration of Drp1 structure．．．．．．．．．．．．．．．．．．14
Figure1-2. The posttranslational modification of Drp1．．．．．．．．．．．．．．．．．．15
Figure1-3. Interaction and co-localization of Drp1/Hydn IV with GSK3beta. ．．．．．．．．．41
Figure1-4. Drp1 is phosphorylated by GSK3beta at Ser693．．．．．．．．．．．．．．．44
Figure1-5. Yeast two-hybrid assay identifying Drp1 inter-/intra- interaction and residue responsible
for GSK3beta-Drp1 binding．．．．．．．．．．．．．．．．．．．．．．．．46
Figure1-6. GTPase hydrolysis activity of Drp1mutants．．．．．．．．．．．．．．．．．．48
Figure1-7. Mitochondrial dynamics of HeLa cells with Drp1 wt and mutants．．．．．．．．．49
Figure1-8. Mitochondrial morphology of HeLa cells with Drp1 wt and K679A mutant ．．．．51
Figure1-9. Mitochondrial dynamics of HeLa cells with Drp1 wt and mutants and were treated with
LiCl and H89．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．52
Figure1-10. Mitochondrial dynamics of HeLa cells with Drp1 wt and mutants treated with H2O2．55
Figure1-11.Overexpressed Drp1 S693D can protect against H2O2-induced mitochondrial
fragmentation and ensuing apoptosis but does not induce autophagy．．．．．．．．57
Figure1-12.Overexpressed Drp1 S693D can protect HEK293 against H2O2-induced mitochondrial
fragmentation and ensuing apoptosis but does not induce autophagy．．．．．．．．60
Figure1-13. Model represents both GSK3β- and PKA-mediated Drp1 phosphorylation induction of
mitochondrial elongation which subsequently causes acquired resistance to H2O2-induced
apoptosis rather than inducing autophagy．．．．．．．．．．．．．．．．．．61
Figure2-1. The Centrosome structure．．．．．．．．．．．．．．．．．．．．．．．．．73
Figure2-2. Bora regulation of Plk1–Aurora A interplay．．．．．．．．．．．．．．．．．．74
Figure2-3. Interaction of AIBp with PLK1 and Aurora-A．．．．．．．．．．．．．．．．．96
Figure2-4. Interaction of AIBp with PLK1, Aurora-A and hNinein.．．．．．．．．．．．．．97
Figure2-5. AIBp, PLK1, and Aurora-A co-localize to the spindle poles in early mitosis．．．．．98
Figure2-6. AIBp and PLK1 co-localize during mitosis.．．．．．．．．．．．．．．．．．99
Figure2-7. AIBp is a substrate of PLK1.．．．．．．．．．．．．．．．．．．．．．．．100
Figure2-8. AIBp is a positive regulator of both Aurora A and PLK1 but a negative regulator blocking phosphorylation of hNinein mediated by Aurora-A and PLK1．．．．．．．．．．．101
Figure2-9. AIBp promotes Aurora-A-mediated Plk1 activation．．．．．．．．．．．．．．．102
Figure2-10. Knockdown of hNinein prevented AIBp centrosome targeting, whereas depletion of AIBp did not affect the localization of hNinein or microtubule nucleation．．．．．．．．104
Figure2-11. AIBp depletion decreased phosphorylation of PLK1 Thr-210 and Aurora-A Thr-288．106
Figure2-12. Depletion of AIBp caused formation of multipolar spindle poles, asymmetrical spindle poles, donut-shaped chromosomes, and chromosome misalignment．．．．．．．．109
Figure2-13. Depletion of AIBp caused mis-localization of TACC3 and ch-TOG but had no effect on the localization of CEP192 and CEP215．．．．．．．．．．．．．．．．．．．111
Figure2-14. The interplay of hNinein, AIBp, Aurora-A, and Plk1 in centrosomal maturation and spindle pole organization．．．．．．．．．．．．．．．．．．．．．．．．．114

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